Cosmetic compositions and methods

ABSTRACT

A method for reducing a presence of sebum on skin, reducing an appearance of shiny skin, or reducing an appearance of the size of skin pores is disclosed. The method can include topically applying to skin a composition comprising a navy bean extract comprising amino acids, an extract of Bambusa vulgaris wendl shoots, and charcoal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/280,467, filed Feb. 20, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/606,421 (U.S. Pat. No. 10,251,832), filed May26, 2017. The contents of the referenced application are incorporatedinto the present application by reference.

BACKGROUND OF THE INVENTION A. Field of the Invention

The current disclosure relates generally to compositions that can beused in cosmetic compositions, used to clean, condition, and/or protectskin, and/or to improve the skin's condition and/or visual appearance.

B. Background

Skin cells are very sensitive and can get affected even by smalldisorders. Usual disorders cause dryness at first and gradually thisleads to multiple problems. The mammalian skin is comprised of sebaceousglands. These glands secrete oily substance called sebum. Sebum iscomposed of fats that do not get dissolved in water. The moisture needsto be retained in the skin and hence, the sebum helps in trapping therequired water in the skin cells. For different age groups, thecomposition of sebum varies too.

Sebum production can be increased by environmental and natural orirregular processes occurring within the human body, such as hormonaland diet changes. Excess sebum may lead to clogged pores that can createa hypoxic environment that supports the growth of acne-causing bacteria.Research has also found that sebum itself can lead to increasedinflammation. Byproducts of the sebum metabolism may cause theaccumulation of molecules that trigger an inflammatory immune response,which can appear as red blotchy spots on the face. In sum, anoverproduction of sebum leads to skin disorders such as acne, pimples,etc., can cause redness due to an inflammatory response, and can clogand enlarge pore size.

The challenge is to remove the excess sebum from the skin whilemaintaining moisture necessary for healthy looking skin. Previousattempts to improve the visual appearance of skin with known skinactive-ingredients have been shown to have various drawbacks such asskin irritation, prolonged recovery periods, or in efficient delivery ofthe promised skin benefits. Current products may not address each ofthese shortcomings.

SUMMARY OF THE INVENTION

The inventors have identified a solution to the problems associated withcurrent cosmetic products. The solution resides in a combination ofingredients including a composition comprising amino acids extractedfrom the navy bean plant, a combination of the extract of Bambusavulgaris wendl shoots and charcoal, Lonicera japonica leaf extract, andsalicylic acid. This combination of components has been shown to beuseful as a cleansing composition that is effective for reducing sebum,increasing clarity of the skin, reducing uneven skin tone and irregularpigmentation, and improving the appearance of the skin by reducing shineand pore size. In some instances, the combination of ingredientsprovides moisture to the skin.

Aspects of the disclosure relate to a composition comprising any one of,any combination of, or all of A composition comprising amino acidsextracted from the navy bean plant, a combination of the extract ofBambusa vulgaris wendl shoots and charcoal, Lonicera japonica leafextract, and salicylic acid. In some embodiments, the composition is anemulsion, a lotion, a gel, or an ointment. In some embodiments, thecomposition comprises a gel. The amounts of the ingredients within thecomposition can vary (e.g., amounts can be as low as 0.000001% to ashigh as 80% w/w or any range therein). In some embodiments, thecomposition comprises 0.05 to 0.5% by weight of amino acids extractedfrom the navy bean plant, 0.0005-0.05% by weight of a combination of theextract of Bambusa vulgaris wendl shoots and charcoal, 0.1-1.1% byweight of Lonicera japonica leaf extract, and 0.1-2.0% by weight ofsalicylic acid. In some embodiments, the composition further comprises asolvent. In some embodiments, the solvent comprises water. In someembodiments, the composition further comprises kaolin, magnesiumaluminum silicate, xanthan gum, and bentonite. Alternatively or inaddition, one or any combination said ingredients can be used in thecompositions described herein. In some embodiments, the compositioncomprises 20-35% by weight kaolin, 1-10% by weight magnesium aluminumsilicate, 0.1-1% by weight of xanthan gum, and 1-8% by weight bentonite.In some embodiments, the composition further comprises one or moreadditional ingredients selected from one or more emulsifiers,moisturizing agents, colorants, preservatives, pH adjusters, thickeningagents, chelating agents, skin conditioning agents, and fragrances.

Further aspects of the disclosure relate to a method of cleansing theskin comprising applying a composition of the disclosure to the skinfollowed by rinsing said composition from the skin after application.Further aspects relate to a method for reducing the presence of sebum,reducing the appearance of shine, and/or reducing the appearance of poresize, the method comprising applying a composition of the disclosure tothe skin followed by rinsing said composition from the skin afterapplication. Further aspects relate to a method for reducing uneven skintone, discrete pigment, and/or mottled pigment, the method comprisingapplying a composition of the disclosure to the skin followed by rinsingsaid composition from the skin after application. In some embodiments,discrete pigment comprises age spots, dark spots, and/or freckles. Insome embodiments, mottled pigment comprises irregular dark blotches. Insome embodiments, uneven skin tone comprises irregular dark blotchesand/or irregular pigmentation. Yet further aspects relate to a methodfor improving brightness intensity, pigment variation, and/or contrast,the method comprising applying a composition of the disclosure to theskin followed by rinsing said composition from the skin afterapplication. In some embodiments, the composition is contacted with theskin for at least about, at most about, or exactly 1, 2, 4, 6, 8, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, or 60, minutes, or any rangederivable therein. In some embodiments of the disclosed aspects, thecomposition is contacted with the skin for at least 2 minutes. In someembodiments, the composition is contacted with the skin for at least 10minutes. In some embodiments, the composition is contacted with the skinfor at least about, at most about, or exactly 2, 4, 6, 8, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, or 1.5, 2, or 3 hours, or any rangederivable therein. In some embodiments, the composition is applied tothe face. In some embodiments, the composition is applied to the neck,chest, arms, hands, torso, or legs. In some embodiments, the methodcomprises applying the composition to the skin at least one time a day.In some embodiments, the method comprises applying the composition tothe skin at least about, at most about, or exactly 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 times a day. In some embodiments, the method comprises dailyapplication for a period of at least one week. In some embodiments, themethod comprises daily application for a period of at least about, atmost about, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 18, 20, 22, 24, 26, 28 days or 5, 6, 7, 8, 9, 10, 12, 14, 16,18, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, weeks or 1, 2, 3, 4, 5 or 6years or more (or any derivable range therein).

In particular aspects, the compositions of the present disclosure areformulated as a topical skin composition. The composition can have adermatologically acceptable vehicle or carrier for the compounds andextracts. The composition can further include a moisturizing agent or ahumectant, a surfactant, a silicone containing compounds, a UV agent, anoil, and/or other ingredients identified in this specification or thoseknown in the art. The composition can be a mask, lotion, cream, gel,serum, emulsion (e.g., oil-in-water, water-in-oil, silicone-in-water,water-in-silicone, water-in-oil-in-water, oil-in-water-in-oil,oil-in-water-in-silicone, etc.), solutions (e.g., aqueous orhydro-alcoholic solutions), anhydrous bases (e.g., lipstick or apowder), ointments, milk, paste, aerosol, solid forms, eye jellies, gelserums, gel emulsions, etc. In other aspects of the present disclosure,compositions can be storage stable or color stable, or both. It is alsocontemplated that the viscosity of the composition can be selected toachieve a desired result, e.g., depending on the type of compositiondesired, the viscosity of such composition can be from about 1 cps towell over 1 million cps or any range or integer derivable therein (e.g.,2 cps, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000,6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000,80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000,800000, 900000, 1000000, 2000000, 3000000, 4000000, 5000000, 10000000,cps, etc., as measured on a Brookfield Viscometer using a TC spindle at2.5 rpm at 25° C.).

The compositions in non-limiting aspects can have a pH of about 6 toabout 9. In other aspects, the pH can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or 14. The compositions can also include preservatives.Non-limiting examples of preservatives include methylparaben,propylparaben, or a mixture of methylparaben and propylparaben. In someembodiments, the composition is paraben-free.

Compositions of the present disclosure can have UVA and UVB absorptionproperties. The compositions can have an sun protection factor (SPF) of2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, or more, or any integer or derivative therein. Thecompositions can be sunscreen lotions, sprays, or creams.

The compositions of the present disclosure can also include any one of,any combination of, or all of the following additional ingredients:water, a chelating agent, a moisturizing agent, a preservative, athickening agent, a silicone containing compound, an essential oil, astructuring agent, a vitamin, a pharmaceutical ingredient, or anantioxidant, or any combination of such ingredients or mixtures of suchingredients. In certain aspects, the composition can include at leasttwo, three, four, five, six, seven, eight, nine, ten, or all of theseadditional ingredients identified in the previous sentence. Non-limitingexamples of these additional ingredients are identified throughout thisspecification and are incorporated into this section by reference. Theamounts of such ingredients can range from 0.0001% to 99.9% by weight orvolume of the composition, or any integer or range in between asdisclosed in other sections of this specification, which areincorporated into this paragraph by reference.

Kits that include the compositions of the present disclosure are alsocontemplated. In certain embodiments, the composition is comprised in acontainer. The container can be a bottle, dispenser, or package. Thecontainer can dispense a pre-determined amount of the composition. Incertain aspects, the compositions is dispensed in a spray, mist, dollop,or liquid. The container can include indicia on its surface. The indiciacan be a word, an abbreviation, a picture, or a symbol.

It is also contemplated that the compositions disclosed throughout thisspecification can be used as a leave-on or rinse-off composition. By wayof example, a leave-on composition can be one that is topically appliedto skin and remains on the skin for a period of time (e.g., at least 5,6, 7, 8, 9, 10, 20, or 30 minutes, or at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours,or overnight or throughout the day). Alternatively, a rinse-offcomposition can be a product that is intended to be applied to the skinand then removed or rinsed from the skin (e.g., with water) within aperiod of time such as less than 5, 4, 3, 2, or 1 minute. An example ofa rinse of composition can be a skin cleanser, shampoo, conditioner, orsoap. An example of a leave-on composition can be a skin moisturizer,sunscreen, mask, overnight cream, or a day cream.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of thedisclosure, and vice versa. Furthermore, compositions of the disclosurecan be used to achieve methods of the disclosure.

In one embodiment, compositions of the present disclosure can bepharmaceutically or cosmetically elegant or can have pleasant tactileproperties. “Pharmaceutically elegant,” “cosmetically elegant,” and/or“pleasant tactile properties” describes a composition that hasparticular tactile properties which feel pleasant on the skin (e.g.,compositions that foam when applied, compositions that are not toowatery or greasy, compositions that have a silky texture, compositionsthat are non-tacky or sticky, etc.). Pharmaceutically or cosmeticallyelegant can also relate to the creaminess or lubricity properties of thecomposition or to the moisture retaining properties of the composition.

Also contemplated is a product comprising a composition of the presentdisclosure. In non-limiting aspects, the product can be a cosmeticproduct. The cosmetic product can be those described in other sectionsof this specification or those known to a person of skill in the art.Non-limiting examples of products include a moisturizer, a cream, alotion, a skin softener, a gel, a wash, a foundation, a night cream, alipstick, a cleanser, a toner, a sunscreen, a mask, an anti-agingproduct, a deodorant, an antiperspirant, a perfume, a cologne, etc.

“Topical application” means to apply or spread a composition onto thesurface of lips or keratinous tissue. “Topical skin composition”includes compositions suitable for topical application on skin and/orkeratinous tissue. Such compositions are typicallydermatologically-acceptable in that they do not have undue toxicity,incompatibility, instability, allergic response, and the like, whenapplied to skin and/or keratinous tissue. Topical skin care compositionsof the present disclosure can have a selected viscosity to avoidsignificant dripping or pooling after application to skin and/orkeratinous tissue.

“Keratinous tissue” includes keratin-containing layers disposed as theoutermost protective covering of mammals and includes, but is notlimited to, lips, skin, hair, and nails.

The term “about” or “approximately” are defined as being close to asunderstood by one of ordinary skill in the art. In one non-limitingembodiment the terms are defined to be within 10%, preferably within 5%,more preferably within 1%, and most preferably within 0.5%.

The term “substantially” and its variations are refers to ranges within10%, within 5%, within 1%, or within 0.5%.

The terms “inhibiting” or “reducing” or any variation of these termsincludes any measurable decrease or complete inhibition to achieve adesired result. The terms “promote” or “increase” or any variation ofthese terms includes any measurable increase or production of a proteinor molecule (e.g., matrix proteins such as fibronectin, laminin,collagen, or elastin or molecules such as hyaluronic acid) to achieve adesired result.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult.

The use of the word “a” or “an” when used in conjunction with the terms“comprising,” “including,” “having,” or “containing,” or any variationsof these terms, in the claims and/or the specification may mean “one,”but it is also consistent with the meaning of “one or more,” “at leastone,” and “one or more than one.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The compositions and methods for their use can “comprise,” “consistessentially of,” or “consist of” any of the ingredients or stepsdisclosed throughout the specification. With respect to the phrase“consisting essentially of,” a basic and novel property of thecompositions and methods of the present disclosure is the ability toproduce a cleansing composition when applied to skin.

Other objects, features, and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the examples,while indicating specific embodiments of the invention, are given by wayof illustration only. Additionally, it is contemplated that changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

A particular composition of the present disclosure is designed to workas a topical composition. The composition relies on a unique combinationof any one of, any combination of, or all of amino acids extracted fromthe navy bean plant, a combination of the extract of Bambusa vulgariswendl shoots and charcoal, Lonicera japonica leaf extract, and salicylicacid. A non-limiting example of such a composition is provided inExample 1, Table 1.

The compositions disclosed herein can be applied to the skin and remainon the skin for a period of time (e.g., at least 1, 2, 3, 4, 5, 10, 20,30, or 60 minutes or more). After which, the composition, if needed, canbe rinsed from the skin or peeled from the skin.

These and other non-limiting aspects of the present disclosure aredescribed in the following sections.

A. Active Ingredients

The present disclosure is premised on a determination that a combinationof active ingredients—amino acids extracted from the navy bean plant, acombination of the extract of Bambusa vulgaris wendl shoots andcharcoal, Lonicera japonica leaf extract, and salicylic acid—can be usedto create a face cleansing composition useful for reducing sebum,increasing clarity of the skin, reducing uneven skin tone and irregularpigmentation, and improving the appearance of the skin by reducing shineand pore size.

This combination of ingredients can be used in different products totreat various skin conditions as described herein. By way ofnon-limiting examples, the combination of ingredients can be formulatedin a mask, emulsion, a gel, a gel emulsion, a gel serum, a lotion, or abody butter.

Amino acids are biological organic compounds composed of amine andcarboxylic acid functional groups, along with a side-chain specific toeach amino acid. The amino acids extracted from the navy bean may beextracted by a variety of different methods that are known in the artand/or described herein.

The compositions of the disclosure comprise a combination of the extractof Bambusa vulgaris wendl shoots and charcoal prepared in glycerin andwater. The extract imparts antioxidant properties as well as absorbanceto the composition.

Lonicera japonica (honeysuckle) leaf extract is an extract of the leavesof the honeysuckle. Honeysuckle contains antiviral, antibacterial, andanti-inflammatory constituents and has a multitude of uses. The extractis also useful as a skin conditioner in the compositions of thedisclosure.

Salicylic acid is a monohydroxybenzoic acid, a type of phenolic acid,and a beta hydroxy acid. This colorless crystalline organic acid iswidely used in organic synthesis and functions as a plant hormone.Salicylic acid is useful for reducing and clearing acne from the skinand provides anti-acne activity to the compositions of the disclosure.

The extracts described herein can be extracts made through extractionmethods known in the art and combinations thereof. Non-limiting examplesof extraction methods include the use of liquid-liquid extraction, solidphase extraction, aqueous extraction, ethyl acetate, alcohol, acetone,oil, supercritical carbon dioxide, heat, pressure, pressure dropextraction, ultrasonic extraction, etc. Extracts can be a liquid, solid,dried liquid, re-suspended solid, etc.

B. Amounts of Ingredients

It is contemplated that the compositions of the present disclosure caninclude any amount of the ingredients discussed in this specification.The compositions can also include any number of combinations ofadditional ingredients described throughout this specification (e.g.,pigments, or additional cosmetic or pharmaceutical ingredients). Theconcentrations of the any ingredient within the compositions can vary.In non-limiting embodiments, for example, the compositions can comprise,consisting essentially of, or consist of, in their final form, forexample, at least about 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%,0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.0010%, 0.0011%, 0.0012%, 0.0013%,0.0014%, 0.0015%, 0.0016%, 0.0017%, 0.0018%, 0.0019%, 0.0020%, 0.0021%,0.0022%, 0.0023%, 0.0024%, 0.0025%, 0.0026%, 0.0027%, 0.0028%, 0.0029%,0.0030%, 0.0031%, 0.0032%, 0.0033%, 0.0034%, 0.0035%, 0.0036%, 0.0037%,0.0038%, 0.0039%, 0.0040%, 0.0041%, 0.0042%, 0.0043%, 0.0044%, 0.0045%,0.0046%, 0.0047%, 0.0048%, 0.0049%, 0.0050%, 0.0051%, 0.0052%, 0.0053%,0.0054%, 0.0055%, 0.0056%, 0.0057%, 0.0058%, 0.0059%, 0.0060%, 0.0061%,0.0062%, 0.0063%, 0.0064%, 0.0065%, 0.0066%, 0.0067%, 0.0068%, 0.0069%,0.0070%, 0.0071%, 0.0072%, 0.0073%, 0.0074%, 0.0075%, 0.0076%, 0.0077%,0.0078%, 0.0079%, 0.0080%, 0.0081%, 0.0082%, 0.0083%, 0.0084%, 0.0085%,0.0086%, 0.0087%, 0.0088%, 0.0089%, 0.0090%, 0.0091%, 0.0092%, 0.0093%,0.0094%, 0.0095%, 0.0096%, 0.0097%, 0.0098%, 0.0099%, 0.0100%, 0.0200%,0.0250%, 0.0275%, 0.0300%, 0.0325%, 0.0350%, 0.0375%, 0.0400%, 0.0425%,0.0450%, 0.0475%, 0.0500%, 0.0525%, 0.0550%, 0.0575%, 0.0600%, 0.0625%,0.0650%, 0.0675%, 0.0700%, 0.0725%, 0.0750%, 0.0775%, 0.0800%, 0.0825%,0.0850%, 0.0875%, 0.0900%, 0.0925%, 0.0950%, 0.0975%, 0.1000%, 0.1250%,0.1500%, 0.1750%, 0.2000%, 0.2250%, 0.2500%, 0.2750%, 0.3000%, 0.3250%,0.3500%, 0.3750%, 0.4000%, 0.4250%, 0.4500%, 0.4750%, 0.5000%, 0.5250%,0.0550%, 0.5750%, 0.6000%, 0.6250%, 0.6500%, 0.6750%, 0.7000%, 0.7250%,0.7500%, 0.7750%, 0.8000%, 0.8250%, 0.8500%, 0.8750%, 0.9000%, 0.9250%,0.9500%, 0.9750%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%,1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%,3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%,4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%,5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%,6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%,7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%,9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% or any range derivable therein, of at least one of theingredients that are mentioned throughout the specification and claims.In non-limiting aspects, the percentage can be calculated by weight orvolume of the total composition. A person of ordinary skill in the artwould understand that the 101384459.1 concentrations can vary dependingon the addition, substitution, and/or subtraction of ingredients in agiven composition.

C. Vehicles

The compositions of the present disclosure can include or beincorporated into all types of vehicles and carriers. The vehicle orcarrier can be a pharmaceutically or dermatologically acceptable vehicleor carrier. Non-limiting examples of vehicles or carriers include water,glycerin, alcohol, oil, a silicon containing compound, a siliconecompound, and wax. Variations and other appropriate vehicles will beapparent to the skilled artisan and are appropriate for use in thepresent disclosure. In certain aspects, the concentrations andcombinations of the compounds, ingredients, and agents can be selectedin such a way that the combinations are chemically compatible and do notform complexes which precipitate from the finished product.

D. Structure

The compositions of the present disclosure can be structured orformulated into a variety of different forms. Non-limiting examplesinclude emulsions (e.g., water-in-oil, water-in-oil-in-water,oil-in-water, silicone-in-water, water-in-silicone, oil-in-water-in-oil,oil-in-water-in-silicone emulsions), creams, lotions, solutions (bothaqueous and hydro-alcoholic), anhydrous bases (such as lipsticks andpowders), gels, masks, peels, and ointments. Variations and otherstructures will be apparent to the skilled artisan and are appropriatefor use in the present disclosure.

E. Additional Ingredients

In addition to the combination of ingredients disclosed by theinventors, the compositions can also include additional ingredients suchas cosmetic ingredients and pharmaceutical active ingredients.Non-limiting examples of these additional ingredients are described inthe following subsections.

1. Cosmetic Ingredients

The CTFA International Cosmetic Ingredient Dictionary and Handbook (2004and 2008) describes a wide variety of non-limiting cosmetic ingredientsthat can be used in the context of the present disclosure. Examples ofthese ingredient classes include: fragrance agents (artificial andnatural; e.g., gluconic acid, phenoxyethanol, Mentha viridis leaf(spearmint) extract, and triethanolamine), dyes and colorants (e.g.,Blue 1, Blue 1 Lake, Red 40, titanium dioxide, D&C blue no. 4, D&C greenno. 5, D&C orange no. 4, D&C red no. 17, D&C red no. 33, D&C violet no.2, D&C yellow no. 10, and D&C yellow no. 11, iron oxides), flavoringagents/aroma agents (e.g., Stevia rebaudiana (sweetleaf) extract, andmenthol), adsorbents, lubricants, solvents, moisturizers (including,e.g., emollients, humectants, film formers, occlusive agents, and agentsthat affect the natural moisturization mechanisms of the skin),water-repellants, UV absorbers (physical and chemical absorbers such aspara-aminobenzoic acid (“PABA”) and corresponding PABA derivatives,titanium dioxide, zinc oxide, etc.), essential oils, vitamins (e.g., A,B, C, D, E, and K), trace metals (e.g., zinc, calcium and selenium),anti-irritants (e.g., steroids and non-steroidal anti-inflammatories),botanical extracts (e.g., Aloe vera, chamomile, cucumber extract, Ginkgobiloba, Ginseng, and rosemary), anti-microbial agents, antioxidants(e.g., BHT and tocopherol), chelating agents (e.g., disodium EDTA andtetrasodium EDTA), preservatives (e.g., methylparaben andpropylparaben), pH adjusters (e.g., sodium hydroxide, triethanolamine,and citric acid), absorbents (e.g., aluminum starch octenylsuccinate,kaolin, corn starch, oat starch, cyclodextrin, talc, and zeolite), skinbleaching and lightening agents (e.g., hydroquinone and niacinamidelactate), humectants (e.g., sorbitol, urea, methyl gluceth-20,saccharide isomerate, and mannitol), exfoliants, waterproofing agents(e.g., magnesium/aluminum hydroxide stearate), skin conditioning agents(e.g., aloe extracts, Rosmarinum officinalis (Rosemary) leaf extract,allantoin, bisabolol, ceramides, dimethicone, hyaluronic acid,biosaccharide gum-1, ethylhexylglycerin, pentylene glycol, hydrogenatedpolydecene, octyldodecyl oleate, and dipotassium glycyrrhizate).Non-limiting examples of some of these ingredients are provided in thefollowing subsections.

a. UV Absorption and/or Reflecting Agents

UV absorption and/or reflecting agents that can be used in combinationwith the compositions of the present disclosure include chemical andphysical sunblocks. Non-limiting examples of chemical sunblocks that canbe used include para-aminobenzoic acid (PABA), PABA esters (glycerylPABA, amyldimethyl PABA and octyldimethyl PABA), butyl PABA, ethyl PABA,ethyl dihydroxypropyl PABA, benzophenones (oxybenzone, sulisobenzone,benzophenone, and benzophenone-1 through 12), cinnamates (octylmethoxycinnamate (octinoxate), isoamyl p-methoxycinnamate, octylmethoxycinnamate, cinoxate, diisopropyl methyl cinnamate, DEA-methoxycinnamate,ethyl diisopropylcinnamate, glyceryl octanoate dimethoxycinnamate andethyl methoxycinnamate), cinnamate esters, salicylates (homomethylsalicylate, benzyl salicylate, glycol salicylate, isopropylbenzylsalicylate, etc.), anthranilates, ethyl urocanate, homosalate,octisalate, dibenzoylmethane derivatives (e.g., avobenzone),octocrylene, octyl triazone, digalloyl trioleate, glycerylaminobenzoate, lawsone with dihydroxyacetone, ethylhexyl triazone,dioctyl butamido triazone, benzylidene malonate polysiloxane,terephthalylidene dicamphor sulfonic acid, disodium phenyldibenzimidazole tetrasulfonate, diethylamino hydroxybenzoyl hexylbenzoate, bis diethylamino hydroxybenzoyl benzoate, bisbenzoxazoylphenyl ethylhexylimino triazine, drometrizole trisiloxane,methylene bis-benzotriazolyl tetramethylbutylphenol, andbis-ethylhexyloxyphenol methoxyphenyltriazine, 4-methylbenzylidenecamphor, and isopentyl 4-methoxycinnamate. Non-limiting examples ofphysical sunblocks include, kaolin, talc, petrolatum and metal oxides(e.g., titanium dioxide and zinc oxide).

b. Moisturizing Agents

Non-limiting examples of moisturizing agents that can be used with thecompositions of the present disclosure include amino acids, chondroitinsulfate, diglycerin, erythritol, fructose, glucose, glycerin, glycerolpolymers, glycol, 1,2,6-hexanetriol, honey, hyaluronic acid,hydrogenated honey, hydrogenated starch hydrolysate, inositol, lactitol,maltitol, maltose, mannitol, natural moisturizing factor, PEG-15butanediol, polyglyceryl sorbitol, salts of pyrrolidone carboxylic acid,potassium PCA, propylene glycol, saccharide isomerate, sodiumglucuronate, sodium PCA, sorbitol, sucrose, trehalose, urea, andxylitol.

Other examples include acetylated lanolin, acetylated lanolin alcohol,alanine, algae extract, Aloe barbadensis, Aloe barbadensis extract, Aloebarbadensis gel, Althea officinalis extract, apricot (Prunus armeniaca)kernel oil, arginine, arginine aspartate, Arnica montana extract,aspartic acid, avocado (Persea gratissima) oil, barrier sphingolipids,butyl alcohol, beeswax, behenyl alcohol, beta-sitosterol, birch (Betulaalba) bark extract, borage (Borago officinalis) extract, butcherbroom(Ruscus aculeatus) extract, butylene glycol, Calendula officinalisextract, Calendula officinalis oil, candelilla (Euphorbia cerifera) wax,canola oil, caprylic/capric triglyceride, cardamom (Elettariacardamomum) oil, carnauba (Copernicia cerifera) wax, carrot (Daucuscarota sativa) oil, castor (Ricinus communis) oil, ceramides, ceresin,ceteareth-5, ceteareth-12, ceteareth-20, cetearyl octanoate, ceteth-20,ceteth-24, cetyl acetate, cetyl octanoate, cetyl palmitate, chamomile(Anthemis nobilis) oil, cholesterol, cholesterol esters, cholesterylhydroxystearate, citric acid, clary (Salvia sclarea) oil, cocoa(Theobroma cacao) butter, coco-caprylate/caprate, coconut (Cocosnucifera) oil, collagen, collagen amino acids, corn (Zea mays) oil,fatty acids, decyl oleate, dimethicone copolyol, dimethiconol, dioctyladipate, dioctyl succinate, dipentaerythrityl hexacaprylate/hexacaprate,DNA, erythritol, ethoxydiglycol, ethyl linoleate, Eucalyptus globulusoil, evening primrose (Oenothera biennis) oil, fatty acids, Geraniummaculatum oil, glucosamine, glucose glutamate, glutamic acid,glycereth-26, glycerin, glycerol, glyceryl distearate, glycerylhydroxystearate, glyceryl laurate, glyceryl linoleate, glycerylmyristate, glyceryl oleate, glyceryl stearate, glyceryl stearate SE,glycine, glycol stearate, glycol stearate SE, glycosaminoglycans, grape(Vitis vinifera) seed oil, hazel (Corylus americana) nut oil, hazel(Corylus avellana) nut oil, hexylene glycol, hyaluronic acid, hybridsafflower (Carthamus tinctorius) oil, hydrogenated castor oil,hydrogenated coco-glycerides, hydrogenated coconut oil, hydrogenatedlanolin, hydrogenated lecithin, hydrogenated palm glyceride,hydrogenated palm kernel oil, hydrogenated soybean oil, hydrogenatedtallow glyceride, hydrogenated vegetable oil, hydrolyzed collagen,hydrolyzed elastin, hydrolyzed glycosaminoglycans, hydrolyzed keratin,hydrolyzed soy protein, hydroxylated lanolin, hydroxyproline, isocetylstearate, isocetyl stearoyl stearate, isodecyl oleate, isopropylisostearate, isopropyl lanolate, isopropyl myristate, isopropylpalmitate, isopropyl stearate, isostearamide DEA, isostearic acid,isostearyl lactate, isostearyl neopentanoate, jasmine (Jasminumofficinale) oil, jojoba (Buxus chinensis) oil, kelp, kukui (Aleuritesmoluccana) nut oil, lactamide MEA, laneth-16, laneth-10 acetate,lanolin, lanolin acid, lanolin alcohol, lanolin oil, lanolin wax,lavender (Lavandula angustifolia) oil, lecithin, lemon (Citrus medicalimonum) oil, linoleic acid, linolenic acid, Macadamia ternifolia nutoil, maltitol, Matricaria (Chamomilla recutita) oil, methyl glucosesesquistearate, methylsilanol PCA, mineral oil, mink oil, Mortierellaoil, myristyl lactate, myristyl myristate, myristyl propionate,neopentyl glycol dicaprylate/dicaprate, octyldodecanol, octyldodecylmyristate, octyldodecyl stearoyl stearate, octyl hydroxystearate, octylpalmitate, octyl salicylate, octyl stearate, oleic acid, olive (Oleaeuropaea) oil, orange (Citrus aurantium dulcis) oil, palm (Elaeisguineensis) oil, palmitic acid, pantethine, panthenol, panthenyl ethylether, paraffin, PCA, peach (Prunus persica) kernel oil, peanut (Arachishypogaea) oil, PEG-8 C12-18 ester, PEG-15 cocamine, PEG-150 distearate,PEG-60 glyceryl isostearate, PEG-5 glyceryl stearate, PEG-30 glycerylstearate, PEG-7 hydrogenated castor oil, PEG-40 hydrogenated castor oil,PEG-60 hydrogenated castor oil, PEG-20 methyl glucose sesquistearate,PEG-40 sorbitan peroleate, PEG-5 soy sterol, PEG-10 soy sterol, PEG-2stearate, PEG-8 stearate, PEG-20 stearate, PEG-32 stearate, PEG-40stearate, PEG-50 stearate, PEG-100 stearate, PEG-150 stearate,pentadecalactone, peppermint (Mentha piperita) extract/oil, petrolatum,phospholipids, plankton extract, polyamino sugar condensate,polyglyceryl-3 diisostearate, polyquaternium-24, polysorbate 20,polysorbate 40, polysorbate 60, polysorbate 80, polysorbate 85,potassium myristate, potassium palmitate, propylene glycol, propyleneglycol dicaprylate/dicaprate, propylene glycol dioctanoate, propyleneglycol dipelargonate, propylene glycol laurate, propylene glycolstearate, propylene glycol stearate SE, PVP, pyridoxine dipalmitate,retinol, retinyl palmitate, rice (Oryza sativa) bran oil, RNA, rosemary(Rosmarinus officinalis) oil, rose oil, safflower (Carthamus tinctorius)oil, sage (Salvia officinalis) oil, sandalwood (Santalum album) oil,serine, serum protein, sesame (Sesamum indicum) oil, shea butter(Butyrospermum parkii), silk powder, sodium chondroitin sulfate, sodiumhyaluronate, sodium lactate, sodium palmitate, sodium PCA, sodiumpolyglutamate, soluble collagen, sorbitan laurate, sorbitan oleate,sorbitan palmitate, sorbitan sesquioleate, sorbitan stearate, sorbitol,soybean (Glycine soja) oil, sphingolipids, squalane, squalene,stearamide MEA-stearate, stearic acid, stearoxy dimethicone,stearoxytrimethylsilane, stearyl alcohol, stearyl glycyrrhetinate,stearyl heptanoate, stearyl stearate, sunflower (Helianthus annuus) seedoil, sweet almond (Prunus amygdalus dukis) oil, synthetic beeswax,tocopherol, tocopheryl acetate, tocopheryl linoleate, tribehenin,tridecyl neopentanoate, tridecyl stearate, triethanolamine, tristearin,urea, vegetable oil, water, waxes, wheat (Triticum vulgare) germ oil,triethyl citrate, and ylang ylang (Cananga odorata) oil.

c. Antioxidants

Non-limiting examples of antioxidants that can be used with thecompositions of the present disclosure include acetyl cysteine, ascorbicacid polypeptide, ascorbyl dipalmitate, ascorbyl methylsilanolpectinate, ascorbyl palmitate, ascorbyl stearate, BHA, BHT, t-butylhydroquinone, cysteine, cysteine HCl, diamylhydroquinone,di-t-butylhydroquinone, dicetyl thiodipropionate, dioleyl tocopherylmethylsilanol, disodium ascorbyl sulfate, distearyl thiodipropionate,ditridecyl thiodipropionate, dodecyl gallate, erythorbic acid, esters ofascorbic acid, ethyl ferulate, ferulic acid, gallic acid esters,hydroquinone, isooctyl thioglycolate, kojic acid, magnesium ascorbate,magnesium ascorbyl phosphate, methylsilanol ascorbate, natural botanicalanti-oxidants such as green tea or grape seed extracts,nordihydroguaiaretic acid, octyl gallate, phenylthioglycolic acid,potassium ascorbyl tocopheryl phosphate, potassium sulfite, propylgallate, quinones, rosmarinic acid, sodium ascorbate, sodium bisulfite,sodium erythorbate, sodium metabisulfite, sodium sulfite, superoxidedismutase, sodium thioglycolate, sorbityl furfural, thiodiglycol,thiodiglycolamide, thiodiglycolic acid, thioglycolic acid, thiolacticacid, thiosalicylic acid, tocophereth-5, tocophereth-10, tocophereth-12,tocophereth-18, tocophereth-50, tocopherol, tocophersolan, tocopherylacetate, tocopheryl linoleate, tocopheryl nicotinate, tocopherylsuccinate, and tris(nonylphenyl)phosphite.

d. Structuring Agents

In other non-limiting aspects, the compositions of the presentdisclosure can include a structuring agent. Structuring agent, incertain aspects, assist in providing rheological characteristics to thecomposition to contribute to the composition's stability. In otheraspects, structuring agents can also function as an emulsifier orsurfactant. Non-limiting examples of structuring agents include stearicacid, palmitic acid, stearyl alcohol, cetyl alcohol, behenyl alcohol,stearic acid, palmitic acid, the polyethylene glycol ether of stearylalcohol having an average of about 1 to about 21 ethylene oxide units,the polyethylene glycol ether of cetyl alcohol having an average ofabout 1 to about 5 ethylene oxide units, and mixtures thereof.

e. Emulsifiers

In certain aspects of the present disclosure, the compositions do notinclude an emulsifier. In other aspects, however, the compositions caninclude one or more emulsifiers. Emulsifiers can reduce the interfacialtension between phases and improve the formulation and stability of anemulsion. The emulsifiers can be nonionic, cationic, anionic, andzwitterionic emulsifiers (See McCutcheon's (1986); U.S. Pat. Nos.5,011,681; 4,421,769; 3,755,560). Non-limiting examples include estersof glycerin, esters of propylene glycol, fatty acid esters ofpolyethylene glycol, fatty acid esters of polypropylene glycol, estersof sorbitol, esters of sorbitan anhydrides, carboxylic acid copolymers,esters and ethers of glucose, ethoxylated ethers, ethoxylated alcohols,alkyl phosphates, polyoxyethylene fatty ether phosphates, fatty acidamides, acyl lactylates, soaps, TEA stearate, DEA oleth-3 phosphate,polyethylene glycol 20 sorbitan monolaurate (polysorbate 20),polyethylene glycol 5 soya sterol, steareth-2, steareth-20, steareth-21,ceteareth-20, cetearyl glucoside, cetearyl alcohol, C12-13 pareth-3,PPG-2 methyl glucose ether distearate, PPG-5-ceteth-20,bis-PEG/PPG-20/20 dimethicone, ceteth-10, polysorbate 80, cetylphosphate, potassium cetyl phosphate, diethanolamine cetyl phosphate,polysorbate 60, glyceryl stearate, PEG-100 stearate, arachidyl alcohol,arachidyl glucoside, bentonite, hydroxypropyl cyclodextrin, and mixturesthereof f. Silicone Containing Compounds

In non-limiting aspects, silicone containing compounds include anymember of a family of polymeric products whose molecular backbone ismade up of alternating silicon and oxygen atoms with side groupsattached to the silicon atoms. By varying the —Si—O— chain lengths, sidegroups, and crosslinking, silicones can be synthesized into a widevariety of materials. They can vary in consistency from liquid to gel tosolids.

The silicone containing compounds that can be used in the context of thepresent disclosure include those described in this specification orthose known to a person of ordinary skill in the art. Non-limitingexamples include silicone oils (e.g., volatile and non-volatile oils),gels, and solids. In certain aspects, the silicon containing compoundsincludes a silicone oils such as a polyorganosiloxane. Non-limitingexamples of polyorganosiloxanes include dimethicone, cyclomethicone,polysilicone-11, phenyl trimethicone, trimethylsilylamodimethicone,stearoxytrimethylsilane, or mixtures of these and other organosiloxanematerials in any given ratio in order to achieve the desired consistencyand application characteristics depending upon the intended application(e.g., to a particular area such as the skin, hair, or eyes). A“volatile silicone oil” includes a silicone oil have a low heat ofvaporization, i.e. normally less than about 50 cal per gram of siliconeoil. Non-limiting examples of volatile silicone oils include:cyclomethicones such as Dow Corning 344 Fluid, Dow Corning 345 Fluid,Dow Corning 244 Fluid, and Dow Corning 245 Fluid, Volatile Silicon 7207(Union Carbide Corp., Danbury, Conn.); low viscosity dimethicones, i.e.dimethicones having a viscosity of about 50 cst or less (e.g.,dimethicones such as Dow Corning 200-0.5 cst Fluid). The Dow CorningFluids are available from Dow Corning Corporation, Midland, Mich.Cyclomethicone and dimethicone are described in the Third Edition of theCTFA Cosmetic Ingredient Dictionary (incorporated by reference) ascyclic dimethyl polysiloxane compounds and a mixture of fully methylatedlinear siloxane polymers end-blocked with trimethylsiloxy units,respectively. Other non-limiting volatile silicone oils that can be usedin the context of the present disclosure include those available fromGeneral Electric Co., Silicone Products Div., Waterford, N.Y. and SWSSilicones Div. of Stauffer Chemical Co., Adrian, Mich.

g. Exfoliating Agent

Exfoliating agents include ingredients that remove dead skin cells onthe skin's outer surface. These agents may act through mechanical,chemical, and/or other means. Non-limiting examples of mechanicalexfoliating agents include abrasives such as pumice, silica, cloth,paper, shells, beads, solid crystals, solid polymers, etc. Non-limitingexamples of chemical exfoliating agents include acids and enzymeexfoliants. Acids that can be used as exfoliating agents include, butare not limited to, glycolic acid, lactic acid, citric acid, alphahydroxy acids, beta hydroxy acids, etc. Other exfoliating agents knownto those of skill in the art are also contemplated as being usefulwithin the context of the present disclosure.

h. Essential Oils

Essential oils include oils derived from herbs, flowers, trees, andother plants. Such oils are typically present as tiny droplets betweenthe plant's cells, and can be extracted by several method known to thoseof skill in the art (e.g., steam distilled, enfleurage (i.e., extractionby using fat), maceration, solvent extraction, or mechanical pressing).When these types of oils are exposed to air they tend to evaporate(i.e., a volatile oil). As a result, many essential oils are colorless,but with age they can oxidize and become darker. Essential oils areinsoluble in water and are soluble in alcohol, ether, fixed oils(vegetal), and other organic solvents. Typical physical characteristicsfound in essential oils include boiling points that vary from about 160°to 240° C. and densities ranging from about 0.759 to about 1.096.

Essential oils typically are named by the plant from which the oil isfound. For example, rose oil or peppermint oil are derived from rose orpeppermint plants, respectively. Non-limiting examples of essential oilsthat can be used in the context of the present disclosure include sesameoil, macadamia nut oil, tea tree oil, evening primrose oil, Spanish sageoil, Spanish rosemary oil, coriander oil, thyme oil, pimento berriesoil, rose oil, anise oil, balsam oil, bergamot oil, rosewood oil, cedaroil, chamomile oil, sage oil, clary sage oil, clove oil, cypress oil,eucalyptus oil, fennel oil, sea fennel oil, frankincense oil, geraniumoil, ginger oil, grapefruit oil, jasmine oil, juniper oil, lavender oil,lemon oil, lemongrass oil, lime oil, mandarin oil, marjoram oil, myrrhoil, neroli oil, orange oil, patchouli oil, pepper oil, black pepperoil, petitgrain oil, pine oil, rose otto oil, rosemary oil, sandalwoodoil, spearmint oil, spikenard oil, vetiver oil, wintergreen oil, orylang ylang. Other essential oils known to those of skill in the art arealso contemplated as being useful within the context of the presentdisclosure.

i. Thickening Agents

Thickening agents, including thickener or gelling agents, includesubstances which that can increase the viscosity of a composition.Thickeners includes those that can increase the viscosity of acomposition without substantially modifying the efficacy of the activeingredient within the composition. Thickeners can also increase thestability of the compositions of the present disclosure. In certainaspects of the present disclosure, thickeners include hydrogenatedpolyisobutene, trihydroxystearin, ammonium acryloyldimethyltaurate/vpcopolymer, or a mixture of them.

Non-limiting examples of additional thickening agents that can be usedin the context of the present disclosure include carboxylic acidpolymers, crosslinked polyacrylate polymers, polyacrylamide polymers,polysaccharides, and gums. Examples of carboxylic acid polymers includecrosslinked compounds containing one or more monomers derived fromacrylic acid, substituted acrylic acids, and salts and esters of theseacrylic acids and the substituted acrylic acids, wherein thecrosslinking agent contains two or more carbon-carbon double bonds andis derived from a polyhydric alcohol (see U.S. Pat. Nos. 5,087,445;4,509,949; 2,798,053; CTFA International Cosmetic Ingredient Dictionary,Fourth edition, 1991, pp. 12 and 80). Examples of commercially availablecarboxylic acid polymers include carbomers, which are homopolymers ofacrylic acid crosslinked with allyl ethers of sucrose or pentaerytritol(e.g., Carbopol™ 900 series from B. F. Goodrich).

Non-limiting examples of crosslinked polyacrylate polymers includecationic and nonionic polymers. One example is sodium polyacrylate.Further examples are described in U.S. Pat. Nos. 5,100,660; 4,849,484;4,835,206; 4,628,078; 4,599,379).

Non-limiting examples of polyacrylamide polymers (including nonionicpolyacrylamide polymers including substituted branched or unbranchedpolymers) include polyacrylamide, isoparaffin and laureth-7, multi-blockcopolymers of acrylamides and substituted acrylamides with acrylic acidsand substituted acrylic acids.

Non-limiting examples of polysaccharides include cellulose,carboxymethyl hydroxyethylcellulose, cellulose acetate propionatecarboxylate, hydroxyethylcellulose, hydroxyethyl ethylcellulose,hydroxypropylcellulose, hydroxypropyl methylcellulose, methylhydroxyethylcellulose, microcrystalline cellulose, sodium cellulosesulfate, and mixtures thereof. Another example is an alkyl substitutedcellulose where the hydroxy groups of the cellulose polymer ishydroxyalkylated (preferably hydroxy ethylated or hydroxypropylated) toform a hydroxyalkylated cellulose which is then further modified with aC10-C30 straight chain or branched chain alkyl group through an etherlinkage. Typically these polymers are ethers of C10-C30 straight orbranched chain alcohols with hydroxyalkylcelluloses. Other usefulpolysaccharides include scleroglucans comprising a linear chain of (1-3)linked glucose units with a (1-6) linked glucose every three unit.

Non-limiting examples of gums that can be used with the presentdisclosure include acacia, agar, algin, alginic acid, ammonium alginate,amylopectin, calcium alginate, calcium carrageenan, carnitine,carrageenan, dextrin, gelatin, gellan gum, guar gum, guarhydroxypropyltrimonium chloride, hectorite, hyaluronic acid, hydratedsilica, hydroxypropyl chitosan, hydroxypropyl guar, karaya gum,cellulose gum, kelp, locust bean gum, natto gum, potassium alginate,potassium carrageenan, propylene glycol alginate, sclerotium gum, sodiumcarboxymethyl dextran, sodium carrageenan, tragacanth gum, xanthan gum,and mixtures thereof.

j. Preservatives

Non-limiting examples of preservatives that can be used in the contextof the present disclosure include quaternary ammonium preservatives suchas sodium benzoate, potassium benzoate, potassium sorbate, iodopropynylbutylcarbamate, polyquaternium-1 and benzalkonium halides (e.g.,benzalkonium chloride (“BAC”) and benzalkonium bromide), parabens (e.g.,methylparabens and propylparabens), phenoxyethanol, benzyl alcohol,chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

2. Pharmaceutical Ingredients

Pharmaceutical active agents are also contemplated as being useful withthe compositions of the present disclosure. Non-limiting examples ofpharmaceutical active agents include anti-acne agents, agents used totreat rosacea, analgesics, anesthetics, anorectals, antihistamines,anti-inflammatory agents including non-steroidal anti-inflammatorydrugs, antibiotics, antifungals, antivirals, antimicrobials, anti-canceractives, scabicides, pediculicides, antineoplastics, antiperspirants,antipruritics, antip soriatic agents, anti seborrheic agents,biologically active proteins and peptides, burn treatment agents,cauterizing agents, depigmenting agents, depilatories, diaper rashtreatment agents, enzymes, hair growth stimulants, hair growthretardants including DFMO and its salts and analogs, hemostatics,kerotolytics, canker sore treatment agents, cold sore treatment agents,dental and periodontal treatment agents, photosensitizing actives, skinprotectant/barrier agents, steroids including hormones andcorticosteroids, sunburn treatment agents, sunscreens, transdermalactives, nasal actives, vaginal actives, wart treatment agents, woundtreatment agents, wound healing agents, etc.

F. Kits

Kits are also contemplated as being used in certain aspects of thepresent disclosure. For instance, compositions of the present disclosurecan be included in a kit. A kit can include a container. Containers caninclude a bottle, a metal tube, a laminate tube, a plastic tube, adispenser, a pressurized container, a barrier container, a package, acompartment, a lipstick container, a compact container, cosmetic pansthat can hold cosmetic compositions, or other types of containers suchas injection or blow-molded plastic containers into which thedispersions or compositions or desired bottles, dispensers, or packagesare retained. The kit and/or container can include indicia on itssurface. The indicia, for example, can be a word, a phrase, anabbreviation, a picture, or a symbol.

The containers can dispense a pre-determined amount of the composition.In other embodiments, the container can be squeezed (e.g., metal,laminate, or plastic tube) to dispense a desired amount of thecomposition. The composition can be dispensed as a spray, an aerosol, aliquid, a fluid, or a semi-solid. The containers can have spray, pump,or squeeze mechanisms. A kit can also include instructions for employingthe kit components as well the use of any other compositions included inthe container. Instructions can include an explanation of how to apply,use, and maintain the compositions.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the disclosure. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the disclosure, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe disclosure.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spirit,and scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1

Formulations having the ingredients from Example 1 were prepared astopical skin compositions according to the current disclosure. Theformulation in Tables 1-2 are examples of a topical skin compositionprepared as a facial mask.

TABLE 1{circumflex over ( )} % Concentration Ingredient (by weight)Water 55 Kaolin 27 Magnesium Aluminum Silicate 5 Dipropylene Glycol 4Bentonite 3 Iron Oxides 1 Butylene Glycol 0.5 Phenoxyethanol 0.5Salicylic Acid 0.5 Lonicera japonica 0.3 (Honeysuckle) Leaf ExtractXanthan Gum 0.2 Navy Bean extract of Amino Acids 0.2 Combination of theextract of 0.01 Bambusa vulgaris wendl shoots and charcoal Excipients*q.s. {circumflex over ( )}Formulation can be prepared by mixing theingredients in a beaker under heat 70-75° C. until homogenous.Subsequently, the formulation can be cooled to standing room temperature(20-25° C.). Further, and if desired, additional ingredients can beadded, for example, to modify the rheological properties of thecomposition or ingredients that provide benefits to skin. *Excipientscan be added, for example, to modify the rheological properties of thecomposition. Alternatively, the amount of water can be varied so long asthe amount of water in the composition is at least 60% w/w, andpreferably between 60 to 85% w/w.

TABLE 2{circumflex over ( )} % Concentration Ingredient (by weight)Water 47 Kaolin 30 Glycerin 7 Propylene Glycol 5 Magnesium AluminumSilicate 4 Bentonite 3 Iron Oxides 1.2 Butylene Glycol 0.7Phenoxyethanol 0.6 Salicylic Acid 0.5 Lonicera japonica 0.3(Honeysuckle) Leaf Extract Xanthan Gum 0.4 Navy Bean extract of AminoAcids 0.2 Combination of the extract of 0.01 Bambusa vulgaris wendlshoots and charcoal Excipients* q.s. {circumflex over ( )}Formulationcan be prepared by mixing the ingredients in a beaker under heat 70-75°C. until homogenous. Subsequently, the formulation can be cooled tostanding room temperature (20-25° C.). Further, and if desired,additional ingredients can be added, for example, to modify therheological properties of the composition or ingredients that providebenefits to skin. *Excipients can be added, for example, to modify therheological properties of the composition. Alternatively, the amount ofwater can be varied so long as the amount of water in the composition isat least 60% w/w, and preferably between 60 to 85% w/w.

Example 2 Sebum Removal and Improved Appearance of Skin afterApplication of Compositions of the Disclosure

The objective of this example was to determine the sebum removalefficacy of the composition of Example 1 when used by a population ofwomen between the ages of 18 and 55, with oily skin.

Twenty-one females, ranging in age from 24 to 50 years, were enrolledand completed the study.

Prior to test article application, baseline Sebumeter measurements weretaken to ensure that both sides of the forehead had Sebumeter (sebum)values of at least 100 μg/cm². If baseline Sebumeter measurements were100 or greater, the subject was enrolled in the study. Followingbaseline Sebumeter measurements, the subject was clinically assessed forpore size and shine on the left and right sides of their forehead. Afterall baseline measurements were completed, the subject applied the testarticle mask to either the left or right side of the forehead (side ofthe forehead that was treated was rotated with odd subject numbersapplying the test article to the left side, and even subject numbersapplying the test article to the right side of their forehead). The maskwas allowed to dry for at least ten minutes after which both sides ofthe forehead were rinsed with water (care was taken to ensure the maskdid not get washed onto the untreated site). After rinsing of the testsite the entire forehead was patted dry with paper towels. Withinfifteen minutes of test article removal final Sebumeter measurementswere taken and the subjects were clinically assessed for pore size andshine as at the baseline assessment. Following all measurements thesubjects were dismissed from the study.

Twenty-one subjects completed the study. A summary of the baseline andpost-treatment/removal Sebumeter data (units=μg/cm²) from the treatedand untreated sites are shown in Text Table 1, Shine and Pore Size data(in arbitrary units) from the treated and untreated sites, as determinedby a clinical evaluator, are shown in Text Table 2.

TABLE 1.0 Sebumeter Summary Test Code and Descriptive Post AttributeTreatment Site Statistics Baseline Treatment/Removal Sebum (Oil)16008.01 Mean 177.7 19.6 (Treated) Percent −88.96 Change* P-Value**<0.0001 Not Applicable Mean 169.4 150.0 (Untreated - Percent −11.47Water Only) Change* P-Value** 0.1497 P-Value***: <0.0001 Treated vs.Untreated Sites *Percent Change relative to baseline **P-Valuedetermined by using a paired t-Test. Bolded values are consideredsignificant at P < 0.05. ***P-Value determined by using a one factorrepeated measures analysis of variance. Bolded values are consideredsignificant at P < 0.05.

TABLE 2.0 Shine and Pore Size Summary Test Code and Descriptive PostAttribute Treatment Site Statistics Baseline Treatment/Removal Shine16008.01 Mean 6.35 1.00 (Treated) Percent −84.25 Change* P-Value**<0.0001 Not Applicable Mean 6.25 4.93 (Untreated - Percent −21.20 WaterOnly) Change* P-Value** 0.0002 P-Value***: <0.0001 Treated vs. UntreatedSites Pore Size 16008.01 Mean 3.90 2.30 (Treated) Percent −41.03 Change*P-Value** <0.0001 Not Applicable Mean 6.25 4.93 (Untreated - Percent−21.20 Water Only) Change* P-Value** 0.0420 P-Value***: 0.0001 Treatedvs. Untreated Sites *Percent Change relative to baseline **P-Valuedetermined by using a paired t-Test. Bolded values are consideredsignificant at P < 0.05. ***P-Value determined by using a one factorrepeated measures analysis of variance. Bolded values are consideredsignificant at P < 0.05.

The results demonstrate that there was a significant reduction inSebumeter values on the treated sites and no significant change inSebumeter values on the untreated sites. Furthermore, when comparing thetreated to untreated sites, the treated sites had significantly greaterchanges (reduction) in Sebumeter values relative to the untreated sites.With respect to pore size, there was a significant reduction in PoreSize values on both the treated and untreated sites. However, whencomparing the treated to untreated sites, the treated sites hadsignificantly greater changes (reduction) in Pore Size values relativeto the untreated sites. Similarly, there was a significant reduction inShine values on both the treated and untreated sites, but comparing thetreated to untreated sites, the treated sites had significantly greaterchanges (reduction) in Shine values relative to the untreated sites.

In conclusion, under the conditions of this study, the composition ofexample 1 was effective at significantly reducing the presence of sebum,and reducing the appearance of both Shine and Pore Size after a singleapplication.

Example 3 Improving Skin Hyperpigmentation with Moisturizer ComprisingNavy Bean Amino Acids

In this example, moisturizing cream with or without navy bean aminoacids was tested for efficacy in improving skin pigmentation andclarity. Thirty one panelists were enrolled in the study and thirtypanelists completed the study: twenty-one panelists were randomly placedin the treatment group and nine panelists were randomly placed in thecontrol group. One panelist dropped out of the study due to personalreasons. The data was collected at Day 0 and weeks 4, 8, and 12 duringstudy visits. Each study visit was approximately fifteen minutes induration. During these visits, panelists were visually graded by thestudy investigator for uneven skin tone, discrete pigment and mottledpigment using the ‘Global Photo Aging Scale’ from 0 to 9 (0=none,9=severe). The visual grading was followed by taking a set of full facephotographs using the VISIA CR and Clarity Pro imaging systems.Photographs obtained from Clarity Pro were analyzed for pigmentationparameters such as brightness intensity, pigment variation and contrast.

Additionally panelists were instructed on how to use the test product.Panelists were provided with a study diary, usage instructions, the testproduct, SPF 30 sunscreen and a moisturizer to be used as needed.

All data obtained at weeks 4, 8 and 12 were compared to baselinereadings using repeated measures ANOVA on ranks. Statisticalsignificance was considered at p value ≤0.05. All data obtained at weeks4, 8 and 12 from the treatment group were compared to baseline readingsobtained from the control group using a T-test. The results of thesestudies is shown in the tables below:

TABLE 1 Treatment Group Mean Percent Improvement from Baseline[Percentage of Panelists Showing Improvement] 4 weeks 8 weeks 12 weeksSkin Parameters (N = 20) (N = 21) (N = 21) Uneven Skin Tone  15%*  14%* 18%* (overall assessment including dark [60%] [62%] [62%] blotches andpigmentation) Discrete Pigment  7%*  20%*  21%* (age spots, dark spots,freckles) [45%] [52%] [48%] Mottled Pigment NS NS  10%* (large irregulardark blotches) [62%] *Statistically significant at 95% confidenceinterval; NS = Not Significant

TABLE 2 Untreated Group Mean Percent Improvement from Baseline[Percentage of Panelists Showing Improvement] 4 weeks 8 weeks 12 weeksSkin Parameters (N = 10) (N = 9) (N = 9) Uneven Skin Tone  18%* NS NS(overall assessment including dark [40%] blotches and pigmentation)Discrete Pigment NS NS NS (age spots, dark spots, freckles) MottledPigment NS NS NS (large irregular dark blotches) *Statisticallysignificant at 95% confidence interval; NS = Not Significant

TABLE 3 Treated and Untreated Group Mean visual grading scores 4 weeks 8weeks 12 weeks Skin Parameters Treated Untreated Treated UntreatedTreated Untreated Uneven Skin Tone 3.20 2.80 3.33 2.56 3.19 2.56(overall assessment including dark blotches and pigmentation) DiscretePigment 3.05 2.2 2.76 1.67 2.57 1.78 (age spots, dark spots, freckles)Mottled Pigment 3.05 2.2 2.86* 1.56* 2.62 2.00 (large irregular darkblotches) *Statistically significant at 95% confidence interval

There was no statistically significant difference between the treatmentand the control group at weeks 4, 8 and 12 for uneven skin tone anddiscrete pigment. There was a statistically significant difference atweek 8 between the treatment and the control group for mottled pigment.However, there was no statistically significant difference between thetwo groups for mottled pigment at weeks 4 and 12.

Clarity Pro Results

TABLE 4 Treatment Group Mean Percent Improvement from Baseline[Percentage of Panelists Showing Improvement] Pigmentation 4 weeks 8weeks 12 weeks Parameters (N = 20) (N = 20) (N = 19) BrightnessIntensity  3%*  6%*  6%* (indicates lightening effect) [68%] [85%] [95%]Pigment Variation NS  6%*  8%* (Std. deviation of all skin [70%] [84%]pixels in the brown space) Contrast NS  8%*  9%* (Contrast ofneighboring skin pixels [70%] [80%] within a region of interest)*Statistically significant at 95% confidence interval; NS = NotSignificant

TABLE 5 Untreated Group Mean Percent Improvement from Baseline[Percentage of Panelists Showing Improvement] Pigmentation 4 weeks 8weeks 12 weeks Parameters (N = 9) (N = 9) (N = 9) Brightness IntensityNS NS  5%* (indicates lightening effect) [78%] Pigment Variation{2%{circumflex over ( )}}  4%*  6%* (Std. deviation of all skin [33%]   [67%] [78%] pixels in the brown space) Contrast NS NS NS (Contrast ofneighboring skin pixels within a region of interest) *Statisticallysignificant at 95% confidence interval; NS = Not Significant,{circumflex over ( )}Statistically significant worsening compared tobaseline at 95% confidence interval.

TABLE 6 Treated and Untreated Group Mean values for pigmentationparameters 4 weeks (N = 20) 8 weeks (N = 20) 12 weeks (N = 19) SkinParameters Treated Untreated Treated Untreated Treated UntreatedBrightness Intensity 45.86 41.73 47.01 41.45 46.49 42.30 (indicateslightening effect) Pigment Variation 26.85 30.24 24.42 28.12 24.40 27.73(Std. deviation of all skin pixels in the brown space) Contrast 16.5924.16 14.96 22.87 15.37 22.71 (Contrast of neighboring skin pixelswithin a region of interest) *Statistically significant at 95%confidence interval.

There was no statistically significant difference between the treatmentand the control group at weeks 4, 8 and 12 for brightness intensity,pigment variation and contrast.

From the data in tables 1-6 of this example, the following conclusionswere determined: With respect to visual grading, the treated groupshowed significant improvement in uneven skin tone and discrete pigmentat weeks 4, 8 and 12 as compared to baseline. The treated group showedsignificant improvement in mottled pigment at week 12 as compared tobaseline. The untreated group showed no significant improvement inmottled and discrete pigment at weeks 4, 8 and 12 as compared tobaseline. The untreated group showed significant improvement for unevenskin tone at week 4 as compared to baseline.

With respect to clarity, the treated group showed significantimprovement for brightness intensity at weeks 4, 8 and 12 weeks ascompared to baseline. The treated group showed significant improvementfor pigment variation and contrast at weeks 8 and 12 weeks compared tobaseline. The untreated group showed significant improvement forbrightness intensity at week 12 compared to baseline. The untreatedgroup showed significant improvement for pigment variation at weeks 8and 12 compared to baseline. The untreated group showed significantworsening for pigment variation at week 4 as compared to baseline. Theuntreated group showed no significant improvement for pigment contrastat weeks 4, 8 and 12 compared to baseline.

Example 4 Additional Assays

Assays that can be used to determine the efficacy of any one of theingredients or any combination of ingredients or compositions havingsaid combination of ingredients disclosed throughout the specificationand claims can be determined by methods known to those of ordinary skillin the art. The following are non-limiting assays that can be used inthe context of the present disclosure. It should be recognized thatother testing procedures can be used, including, for example, objectiveand subjective procedures.

B16 Pigmentation Assay: Melanogenesis is the process by whichmelanocytes produce melanin, a naturally produced pigment that impartscolor to skin, hair, and eyes. Inhibiting melanogenesis is beneficial toprevent skin darkening and lighten dark spots associated with aging.This bioassay utilizes B16-F1 melanocytes (ATCC), an immortalized mousemelanoma cell line, to analyze the effect of compounds on melanogenesis.The endpoint of this assay is a spectrophotometric measurement ofmelanin production and cellular viability. B16-F1 melanocytes, can becultivated in standard DMEM growth medium with 10% fetal bovine serum(Mediatech) at 37° C. in 10% CO₂ and then treated with any one of theactive ingredients, combination of ingredients, or compositions havingsaid combinations disclosed in the specification for 6 days. Followingincubation, melanin secretion is measured by absorbance at 405 nm andcellular viability is quantified.

Collagen Stimulation Assay: Collagen is an extracellular matrix proteincritical for skin structure. Increased synthesis of collagen helpsimprove skin firmness and elasticity. This bioassay can be used toexamine the effect of any one of the active ingredients, combination ofingredients, or compositions having said combinations disclosed in thespecification on the production of procollagen peptide (a precursor tocollagen) by human epidermal fibroblasts. The endpoint of this assay isa spectrophotometric measurement that reflects the presence ofprocollagen peptide and cellular viability. The assay employs thequantitative sandwich enzyme immunoassay technique whereby a monoclonalantibody specific for procollagen peptide has been pre-coated onto amicroplate. Standards and samples can be pipetted into the wells and anyprocollagen peptide present is bound by the immobilized antibody. Afterwashing away any unbound substances, an enzyme-linked polyclonalantibody specific for procollagen peptide can be added to the wells.Following a wash to remove any unbound antibody-enzyme reagent, asubstrate solution can be added to the wells and color develops inproportion to the amount of procollagen peptide bound in the initialstep using a microplate reader for detection at 450 nm. The colordevelopment can be stopped and the intensity of the color can bemeasured. For generation of samples and controls, subconfluent normalhuman adult epidermal fibroblasts (Cascade Biologics) cultivated instandard DMEM growth medium with 10% fetal bovine serum (Mediatech) at37° C. in 10% CO₂, can be treated with each of the combination ofingredients or compositions having said combinations disclosed in thespecification for 3 days. Following incubation, cell culture medium canbe collected and the amount of procollagen peptide secretion quantifiedusing a sandwich enzyme linked immuno-sorbant assay (ELISA) from Takara(#MK101). Using a composition comprising Lonicera Japonica leaf extract,collagen production was found to be increased by about 80% compared tocontrols.

Elastin Stimulation Assay: Elastin is a connective tissue protein thathelps skin resume shape after stretching or contracting. Elastin is alsoan important load-bearing protein used in places where mechanical energyis required to be stored. Elastin is made by linking many solubletropoelastin protein molecules, in a reaction catalyzed by lysyloxidase. Elastin secretion and elastin fibers can be monitored incultured human fibroblasts by staining of cultured human fibroblastsusing immunofluorescent antibodies directed against elastin.

Laminin and Fibronectin Stimulation Assay: Laminin and fibronectin aremajor proteins in the dermal-epidermal junction (DEJ) (also referred toas the basement membrane). The DEJ is located between the dermis and theepidermis interlocks forming fingerlike projections called rete ridges.The cells of the epidermis receive their nutrients from the bloodvessels in the dermis. The rete ridges increase the surface area of theepidermis that is exposed to these blood vessels and the needednutrients. The DEJ provides adhesion of the two tissue compartments andgoverns the structural integrity of the skin. Laminin and fibronectinare two structural glycoproteins located in the DEJ. Considered the gluethat holds the cells together, laminin and fibronectin are secreted bydermal fibroblasts to help facilitate intra- and inter-cellular adhesionof the epidermal calls to the DEJ. Laminin and fibronectin secretion canbe monitored by quantifying laminin and fibronectin in cell supernatantsof cultured human fibroblasts treated for 3 days with culture mediumwith or without 1.0% final concentration of the test ingredient(s).Following incubation, laminin and fibronectin content can be measuredusing immunofluorescent antibodies directed against each protein in anenzyme linked immuno-sorbant assay (ELISA). Measurements are normalizedfor cellular metabolic activity, as determined by bioconversion of3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS). Using a composition comprising Lonicera Japonica leaf extract,laminin secretion was found to be increased by about 78% compared tocontrols.

Tumor Necrosis Factor Alpha (TNF-α) Assay: The prototype ligand of theTNF superfamily, TNF-α, is a pleiotropic cytokine that plays a centralrole in inflammation. Increase in its expression is associated with anup regulation in pro-inflammatory activity. This bioassay can be used toanalyze the effect of any one of the active ingredients, combination ofingredients, or compositions having said combinations disclosed in thespecification on the production of TNF-α by human epidermalkeratinocytes. The endpoint of this assay can be a spectrophotometricmeasurement that reflects the presence of TNF-α and cellular viability.The assay employs the quantitative sandwich enzyme immunoassay techniquewhereby a monoclonal antibody specific for TNF-α has been pre-coatedonto a microplate. Standards and samples can be pipetted into the wellsand any TNF-α present is bound by the immobilized antibody. Afterwashing away any unbound substances, an enzyme-linked polyclonalantibody specific for TNF-α can be added to the wells. Following a washto remove any unbound antibody-enzyme reagent, a substrate solution canbe added to the wells and color develops in proportion to the amount ofTNF-α bound in the initial step using a microplate reader for detectionat 450 nm. The color development can be stopped and the intensity of thecolor can be measured. Subconfluent normal human adult keratinocytes(Cascade Biologics) cultivated in EpiLife standard growth medium(Cascade Biologics) at 37° C. in 5% CO₂, can be treated with phorbol12-myristate 13-acetate (PMA, 10 ng/ml, Sigma Chemical, #P1585-1MG) andany one of the active ingredients, combination of ingredients, orcompositions having said combinations disclosed in the specification for6 hours. PMA has been shown to cause a dramatic increase in TNF-αsecretion which peaks at 6 hours after treatment. Following incubation,cell culture medium can be collected and the amount of TNF-α secretionquantified using a sandwich enzyme linked immuno-sorbant assay (ELISA)from R&D Systems (#DTA00C).

Antioxidant (AO) Assay: An in vitro bioassay that measures the totalanti-oxidant capacity of any one of the ingredients, combination ofingredients, or compositions having said combinations disclosed in thespecification. The assay relies on the ability of antioxidants in thesample to inhibit the oxidation of ABTS®(2,2′-azino-di-[3-ethylbenzthiazoline sulphonate]) to ABTS®+ bymetmyoglobin. The antioxidant system of living organisms includesenzymes such as superoxide dismutase, catalase, and glutathioneperoxidase; macromolecules such as albumin, ceruloplasmin, and ferritin;and an array of small molecules, including ascorbic acid, α-tocopherol,β-carotene, reduced glutathione, uric acid, and bilirubin. The sum ofendogenous and food-derived antioxidants represents the totalantioxidant activity of the extracellular fluid. Cooperation of all thedifferent antioxidants provides greater protection against attack byreactive oxygen or nitrogen radicals, than any single compound alone.Thus, the overall antioxidant capacity may give more relevant biologicalinformation compared to that obtained by the measurement of individualcomponents, as it considers the cumulative effect of all antioxidantspresent in plasma and body fluids. The capacity of the antioxidants inthe sample to prevent ABTS oxidation is compared with that of Trolox, awater-soluble tocopherol analogue, and is quantified as molar Troloxequivalents. Anti-Oxidant capacity kit #709001 from Cayman Chemical (AnnArbor, Mich. USA) can be used as an in vitro bioassay to measure thetotal anti-oxidant capacity of each of any one of the activeingredients, combination of ingredients, or compositions having saidcombinations disclosed in the specification. The protocol can befollowed according to manufacturer recommendations. A compositioncomprising Lonicera japonica leaf extract was found to inhibit oxidationby 97% compared to controls.

ORAC Assay: Oxygen Radical Absorption (or Absorbance) Capacity (ORAC) ofany one of the active ingredients, combination of ingredients, orcompositions having said combinations disclosed in the specification canalso be assayed by measuring the antioxidant activity of suchingredients or compositions. Antioxidant activity indicates a capabilityto reduce oxidizing agents (oxidants). This assay quantifies the degreeand length of time it takes to inhibit the action of an oxidizing agent,such as oxygen radicals, that are known to cause damage to cells (e.g.,skin cells). The ORAC value of any one of the active ingredients,combination of ingredients, or compositions having said combinationsdisclosed in the specification can be determined by methods known tothose of ordinary skill in the art (see U.S. Publication Nos.2004/0109905 and 2005/0163880; and commercially available kits such asZen-Bio ORAC Anti-oxidant Assay kit (#AOX-2)). The Zen-Bio ORACAnti-oxidant Assay kit measures the loss of fluorescein fluorescenceover time due to the peroxyl-radical formation by the breakdown of AAPH(2,2′-axobis-2-methyl propanimidamide, dihydrochloride). Trolox, a watersoluble vitamin E analog, serves as positive control inhibitionfluorescein decay in a dose dependent manner.

Mushroom tyrosinase activity assay: In mammalian cells, tyrosinasecatalyzes two steps in the multi-step biosynthesis of melanin pigmentsfrom tyrosine (and from the polymerization of dopachrome). Tyrosinase islocalized in melanocytes and produces melanin (aromatic quinonecompounds) that imparts color to skin, hair, and eyes. Purified mushroomtyrosinase (Sigma) can be incubated with its substrate L-Dopa (Fisher)in the presence or absence of each of the active ingredients, any one ofthe combination of ingredients, or compositions having said combinationsdisclosed in the specification. Pigment formation can be evaluated bycolorimetric plate reading at 490 nm. The percent inhibition of mushroomtyrosinase activity can be calculated compared to non-treated controlsto determine the ability of test ingredients or combinations thereof toinhibit the activity of purified enzyme. Test extract inhibition wascompared with that of kojic acid (Sigma). Using this assay, acomposition comprising vegetable amino acids was found to inhibittyrosinase by 39% compared to controls.

Matrix Metalloproteinase 3 and 9 Enzyme Activity (MMP3; MMP9) Assay: Anin vitro matrix metalloprotease (MMP) inhibition assay. MMPs areextracellular proteases that play a role in many normal and diseasestates by virtue of their broad substrate specificity. MMP3 substratesinclude collagens, fibronectins, and laminin; while MMP9 substratesinclude collagen VII, fibronectins and laminin. Using Colorimetric DrugDiscovery kits from BioMol International for MMP3 (AK-400) and MMP-9(AK-410), this assay is designed to measure protease activity of MMPsusing a thiopeptide as a chromogenic substrate(Ac-PLG42-mercapto-4-methyl-pentanoylRG-OC2H5)5,6. The MMP cleavage sitepeptide bond is replaced by a thioester bond in the thiopeptide.Hydrolysis of this bond by an M1VIP produces a sulfhydryl group, whichreacts with DTNB [5,5′-dithiobis(2-nitrobenzoic acid), Ellman's reagent]to form 2-nitro-5-thiobenzoic acid, which can be detected by itsabsorbance at 412 nm (c=13,600 M−1 cm−1 at pH 6.0 and above 7). Theactive ingredients, any one of the combination of ingredients, orcompositions having said combinations disclosed in the specification canbe assayed.

Matrix Metalloproteinase 1 Enzyme Activity (MMP1) Assay: An in vitromatrix metalloprotease (MMP) inhibition assay. MMPs are extracellularproteases that play a role in many normal and disease states by virtueof their broad substrate specificity. MMP1 substrates include collagenIV. The Molecular Probes Enz/Chek Gelatinase/Collagenase Assay kit(#E12055) utilizes a fluorogenic gelatin substrate to detect MMP1protease activity. Upon proteolytic cleavage, bright green fluorescenceis revealed and may be monitored using a fluorescent microplate readerto measure enzymatic activity.

The Enz/Chek Gelatinase/Collagenase Assay kit (#E12055) from Invitrogenis designed as an in vitro assay to measure MMP1 enzymatic activity. Theactive ingredients, any one of the combination of ingredients, orcompositions having said combinations disclosed in the specification canbe assayed. The assay relies upon the ability of purified MMP1 enzyme todegrade a fluorogenic gelatin substrate. Once the substrate isspecifically cleaved by MMP1 bright green fluorescence is revealed andmay be monitored using a fluorescent microplate reader. Test materialsare incubated in the presence or absence of the purified enzyme andsubstrate to determine their protease inhibitor capacity.

Cyclooxygenase (COX) Assay: An in vitro cyclooxygenase-1 and -2 (COX-1,-2) inhibition assay. COX is a bifunctional enzyme exhibiting bothcyclooxygenase and peroxidase activities. The cyclooxygenase activityconverts arachidonic acid to a hydroperoxy endoperoxide (ProstaglandinG2; PGG2) and the peroxidase component reduces the endoperoxide(Prostaglandin H2; PGH2) to the corresponding alcohol, the precursor ofprostaglandins, thromboxanes, and prostacyclins. This COX Inhibitorscreening assay measures the peroxidase component of cyclooxygenases.The peroxidase activity is assayed colorimetrically by monitoring theappearance of oxidized N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD).This inhibitor screening assay includes both COX-1 and COX-2 enzymes inorder to screen isozyme-specific inhibitors. The Colormetric COX (ovine)Inhibitor screening assay (#760111, Cayman Chemical) can be used toanalyze the effects of each of the active ingredients, any one of thecombination of ingredients, or compositions having said combinationsdisclosed in the specification on the activity of purified cyclooxygnaseenzyme (COX-1 or COX-2). According to manufacturer instructions,purified enzyme, heme and test extracts can be mixed in assay buffer andincubated with shaking for 15 min at room temperature. Followingincubation, arachidonic acid and colorimetric substrate can be added toinitiate the reaction. Color progression can be evaluated bycolorimetric plate reading at 590 nm. The percent inhibition of COX-1 orCOX-2 activity can be calculated compared to non-treated controls todetermine the ability of test extracts to inhibit the activity ofpurified enzyme.

Lipoxygenase (LO) Assay: An in vitro lipoxygenase (LO) inhibition assay.LOs are non-heme iron-containing dioxygenases that catalyze the additionof molecular oxygen to fatty acids. Linoleate and arachidonate are themain substrates for LOs in plants and animals. Arachadonic acid may thenbe converted to hydroxyeicosotrienenoic (HETE) acid derivatives, thatare subsequently converted to leukotrienes, potent inflammatorymediators. This assay provides an accurate and convenient method forscreening lipoxygenase inhibitors by measuring the hydroperoxidesgenerated from the incubation of a lipoxygenase (5-, 12-, or 15-LO) witharachidonic acid. The Colorimetric LO Inhibitor screening kit (#760700,Cayman Chemical) can be used to determine the ability of each of theactive ingredients, any one of the combination of ingredients, orcompositions having said combinations disclosed in the specification toinhibit enzyme activity. Purified 15-lipoxygenase and test ingredientscan be mixed in assay buffer and incubated with shaking for 10 min atroom temperature. Following incubation, arachidonic acid can be added toinitiate the reaction and the mixtures can be incubated for anadditional 10 min at room temperature. Colorimetric substrate can beadded to terminate catalysis and color progression can be evaluated byfluorescence plate reading at 490 nm. The percent inhibition oflipoxyganse activity can be calculated compared to non-treated controlsto determine the ability of each of the active ingredients, any one ofthe combination of ingredients, or compositions having said combinationsdisclosed in the specification to inhibit the activity of purifiedenzyme.

Elastase Assay: EnzChek® Elastase Assay (Kit # E-12056) from MolecularProbes (Eugene, Oreg. USA) can be used as an in vitro enzyme inhibitionassay for measuring inhibition of elastase activity for each of theactive ingredients, any one of the combination of ingredients, orcompositions having said combinations disclosed in the specification.The EnzChek kit contains soluble bovine neck ligament elastin that canbe labeled with dye such that the conjugate's fluorescence can bequenched. The non-fluorescent substrate can be digested by elastase orother proteases to yield highly fluorescent fragments. The resultingincrease in fluorescence can be monitored with a fluorescence microplatereader. Digestion products from the elastin substrate have absorptionmaxima at ˜505 nm and fluorescence emission maxima at ˜515 nm. Thepeptide, N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone, can beused as a selective, collective inhibitor of elastase when utilizing theEnzChek Elastase Assay Kit for screening for elastase inhibitors.

Oil Control Assay: An assay to measure reduction of sebum secretion fromsebaceous glands and/or reduction of sebum production from sebaceousglands can be assayed by using standard techniques known to those havingordinary skill in the art. In one instance, the forehead can be used.Each of the active ingredients, any one of the combination ofingredients, or compositions having said combinations disclosed in thespecification can be applied to one portion of the forehead once ortwice daily for a set period of days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or more days), while another portion of the foreheadis not treated with the composition. After the set period of daysexpires, then sebum secretion can be assayed by application of fineblotting paper to the treated and untreated forehead skin. This is doneby first removing any sebum from the treated and untreated areas withmoist and dry cloths. Blotting paper can then be applied to the treatedand untreated areas of the forehead, and an elastic band can be placedaround the forehead to gently press the blotting paper onto the skin.After 2 hours the blotting papers can be removed, allowed to dry andthen transilluminated. Darker blotting paper correlates with more sebumsecretion (or lighter blotting paper correlates with reduced sebumsecretion.

Erythema Assay: An assay to measure the reduction of skin redness can beevaluated using a Minolta Chromometer. Skin erythema may be induced byapplying a 0.2% solution of sodium dodecyl sulfate on the forearm of asubject. The area is protected by an occlusive patch for 24 hrs. After24 hrs, the patch is removed and the irritation-induced redness can beassessed using the a* values of the Minolta Chroma Meter. The a* valuemeasures changes in skin color in the red region. Immediately afterreading, the area is treated with the active ingredients, any one of thecombination of ingredients, or compositions having said combinationsdisclosed in the specification. Repeat measurements can be taken atregular intervals to determine the formula's ability to reduce rednessand irritation.

Skin Moisture/Hydration Assay: Skin moisture/hydration benefits can bemeasured by using impedance measurements with the Nova Dermal PhaseMeter. The impedance meter measures changes in skin moisture content.The outer layer of the skin has distinct electrical properties. Whenskin is dry it conducts electricity very poorly. As it becomes morehydrated increasing conductivity results. Consequently, changes in skinimpedance (related to conductivity) can be used to assess changes inskin hydration. The unit can be calibrated according to instrumentinstructions for each testing day. A notation of temperature andrelative humidity can also be made. Subjects can be evaluated asfollows: prior to measurement they can equilibrate in a room withdefined humidity (e.g., 30-50%) and temperature (e.g., 68-72° C.). Threeseparate impedance readings can be taken on each side of the face,recorded, and averaged. The T5 setting can be used on the impedancemeter which averages the impedance values of every five secondsapplication to the face. Changes can be reported with statisticalvariance and significance. Each of the active ingredients, any one ofthe combination of ingredients, or compositions having said combinationsdisclosed in the specification can be assayed according to this process.

Skin Clarity and Reduction in Freckles and Age Spots Assay: Skin clarityand the reduction in freckles and age spots can be evaluated using aMinolta Chromometer. Changes in skin color can be assessed to determineirritation potential due to product treatment using the a* values of theMinolta Chroma Meter. The a* value measures changes in skin color in thered region. This is used to determine whether each of the activeingredients, any one of the combination of ingredients, or compositionshaving said combinations disclosed in the specification is inducingirritation. The measurements can be made on each side of the face andaveraged, as left and right facial values. Skin clarity can also bemeasured using the Minolta Meter. The measurement is a combination ofthe a*, b, and L values of the Minolta Meter and is related to skinbrightness, and correlates well with skin smoothness and hydration. Skinreading is taken as above. In one non-limiting aspect, skin clarity canbe described as L/C where C is chroma and is defined as (a²+b²)^(1/2).

Skin Dryness, Surface Fine Lines, Skin Smoothness, and Skin Tone Assay:Skin dryness, surface fine lines, skin smoothness, and skin tone can beevaluated with clinical grading techniques. For example, clinicalgrading of skin dryness can be determined by a five point standardKligman Scale: (0) skin is soft and moist; (1) skin appears normal withno visible dryness; (2) skin feels slightly dry to the touch with novisible flaking; (3) skin feels dry, tough, and has a whitish appearancewith some scaling; and (4) skin feels very dry, rough, and has a whitishappearance with scaling. Evaluations can be made independently by twoclinicians and averaged.

Clinical Grading of Skin Tone Assay: Clinical grading of skin tone canbe performed via a ten point analog numerical scale: (10) even skin ofuniform, pinkish brown color. No dark, erythremic, or scaly patches uponexamination with a hand held magnifying lens. Microtexture of the skinvery uniform upon touch; (7) even skin tone observed withoutmagnification. No scaly areas, but slight discolorations either due topigmentation or erythema. No discolorations more than 1 cm in diameter;(4) both skin discoloration and uneven texture easily noticeable. Slightscaliness. Skin rough to the touch in some areas; and (1) uneven skincoloration and texture. Numerous areas of scaliness and discoloration,either hypopigmented, erythremic or dark spots. Large areas of unevencolor more than 1 cm in diameter. Evaluations were made independently bytwo clinicians and averaged.

Clinical Grading of Skin Smoothness Assay: Clinical grading of skinsmoothness can be analyzed via a ten point analog numerical scale: (10)smooth, skin is moist and glistening, no resistance upon dragging fingeracross surface; (7) somewhat smooth, slight resistance; (4) rough,visibly altered, friction upon rubbing; and (1) rough, flaky, unevensurface. Evaluations were made independently by two clinicians andaveraged.

Skin Smoothness and Wrinkle Reduction Assay With Methods Disclosed inPackman et al. (1978): Skin smoothness and wrinkle reduction can also beassessed visually by using the methods disclosed in Packman et al.(1978). For example, at each subject visit, the depth, shallowness andthe total number of superficial facial lines (SFLs) of each subject canbe carefully scored and recorded. A numerical score was obtained bymultiplying a number factor times a depth/width/length factor. Scoresare obtained for the eye area and mouth area (left and right sides) andadded together as the total wrinkle score.

Skin Firmness Assay with a Hargens Ballistometer: Skin firmness can bemeasured using a Hargens ballistometer, a device that evaluates theelasticity and firmness of the skin by dropping a small body onto theskin and recording its first two rebound peaks. The ballistometry is asmall lightweight probe with a relatively blunt tip (4 square mm-contactarea) was used. The probe penetrates slightly into the skin and resultsin measurements that are dependent upon the properties of the outerlayers of the skin, including the stratum corneum and outer epidermisand some of the dermal layers.

Skin Softness/Suppleness Assay with a Gas Bearing Electrodynamometer:Skin softness/suppleness can be evaluated using the Gas BearingElectrodynamometer, an instrument that measures the stress/strainproperties of the skin. The viscoelastic properties of skin correlatewith skin moisturization. Measurements can be obtained on thepredetermined site on the cheek area by attaching the probe to the skinsurface with double-stick tape. A force of approximately 3.5 gm can beapplied parallel to the skin surface and the skin displacement isaccurately measured. Skin suppleness can then be calculated and isexpressed as DSR (Dynamic Spring Rate in gm/mm).

Appearance of Lines and Wrinkles Assay with Replicas: The appearance oflines and wrinkles on the skin can be evaluated using replicas, which isthe impression of the skin's surface. Silicone rubber like material canbe used. The replica can be analyzed by image analysis. Changes in thevisibility of lines and wrinkles can be objectively quantified via thetaking of silicon replicas form the subjects' face and analyzing thereplicas image using a computer image analysis system. Replicas can betaken from the eye area and the neck area, and photographed with adigital camera using a low angle incidence lighting. The digital imagescan be analyzed with an image processing program and are of the replicascovered by wrinkles or fine lines was determined.

Surface Contour of the Skin Assay with a Profilometer/Stylus Method: Thesurface contour of the skin can be measured by using theprofilometer/Stylus method. This includes either shining a light ordragging a stylus across the replica surface. The vertical displacementof the stylus can be fed into a computer via a distance transducer, andafter scanning a fixed length of replica a cross-sectional analysis ofskin profile can be generated as a two-dimensional curve. This scan canbe repeated any number of times along a fix axis to generate a simulated3-D picture of the skin. Ten random sections of the replicas using thestylus technique can be obtained and combined to generate averagevalues. The values of interest include Ra which is the arithmetic meanof all roughness (height) values computed by integrating the profileheight relative to the mean profile height. Rt which is the maximumvertical distance between the highest peak and lowest trough, and Rzwhich is the mean peak amplitude minus the mean peak height. Values aregiven as a calibrated value in mm. Equipment should be standardizedprior to each use by scanning metal standards of know values. Ra Valuecan be computed by the following equation: R_(a)=Standardize roughness;l_(m)=the traverse (scan) length; and y=the absolute value of thelocation of the profile relative to the mean profile height (x-axis).

MELANODERM™ Assay: In other non-limiting aspects, the efficacy of eachof the active ingredients, any one of the combination of ingredients, orcompositions having said combinations disclosed in the specification canbe evaluated by using a skin analog, such as, for example, MELANODERM™.Melanocytes, one of the cells in the skin analog, stain positively whenexposed to L-dihydroxyphenyl alanine (L-DOPA), a precursor of melanin.The skin analog, MELANODERM™, can be treated with a variety of basescontaining each of the active ingredients, any one of the combination ofingredients, or compositions having said combinations disclosed in thespecification or with the base alone as a control. Alternatively, anuntreated sample of the skin analog can be used as a control.

Production of Filaggrin: Changes in the production of filaggrin inkeratinocytes due to each of the active ingredients, any one of thecombination of ingredients, or compositions having said combinationsdisclosed in the specification can be measured. Filaggrin is theprecursor to Natural Moisturizing Factor (NMF) in the skin. IncreasedNMF increases the moisture content of the skin. Filaggrin production intreated and non-treated keratinocytes can be determined using a bioassaythat analyzes filaggrin concentration in keratinocyte cell lysates. Anon-limiting example of a bioassay that can be used to quantifyfilaggrin production is the PROTEINSIMPLE® Simon™ western blottingprotocol. For each sample, normal human epidermal keratinocytes (NHEK)are grown in EPI-200—Mattek Epilife® growth media with calcium from LifeTechnologies (M-EP-500-CA). NHEK are incubated in growth mediumovernight at 37° C. in 5% CO₂ prior to treatment. NHEK are thenincubated in growth medium with 1% test compound/extract or nocompound/extract (negative control) for 24 to 36 hours. The NHEK canthen be washed, collected, and stored on ice or colder until lysed onice using a lysis buffer and sonication. The protein concentrations ofthe samples can be determined and used to normalize the samples. Thelysates can be stored at −80° C. until use in the quantification assay.

The PROTEINSIMPLE® Simon™ western blotting bioassay assay employs aquantitative western blotting immunoassay technique using an antibodyspecific for filaggrin to quantitatively detect filaggrin in the testsamples. Cell samples are lysed and normalized for proteinconcentration. Normalized samples and molecular weight standards canthen be loaded and ran on a denatured protein separation gel usingcapillary electrophoresis. The proteins in the gel are immobilized andimmunoprobed using a primary antibody specific for filaggrin. Theimmobilized proteins can then be immunoprobed with an enzyme-linkeddetection antibody that binds the primary antibody. A chemiluminescentsubstrate solution can then be added to the immobilized proteins toallow chemiluminescent development in proportion to the amount offilaggrin bound in the immobilization. The chemiluminescent developmentis stopped at a specific time and the intensity of the chemiluminescentsignal can be measured and compared to positive and negative controls.

Production of Occludin: Changes in the production of occludin inkeratinocytes due to each of the active ingredients, any one of thecombination of ingredients, or compositions having said combinationsdisclosed in the specification can be measured. Occludin is a proteincritical to the formulation of tight junctions and the skin's moisturebarrier function. A non-limiting example of how occludin production intreated and non-treated keratinocytes can be determined is by the use ofa bioassay that analyzes occludin concentration in keratinocyte celllysates. The bioassay can be performed using PROTEINSIMPLE® Simon™western blotting protocol. For the samples, adult human epidermalkeratinocytes (HEKa) from Life Technologies (C-005-5C) can be grown at37° C. and 5% CO₂ for 24 hours in Epilife growth media with calcium fromLife Technologies (M-EP-500-CA) supplemented with Keratinocyte GrowthSupplement (HKGS) from Life Technologies (S-101-5). HEKa are thenincubated in growth medium with test compound/extract, nocompound/extract for negative control, or with 1 mM CaCl₂) for positivecontrol for 24 to 48 hours. The HEKa are then washed, collected, andstored on ice or colder until lysed on ice using a lysis buffer andsonication. The protein concentrations of the samples can be determinedand used to normalize the samples. The lysates are stored at −80° C.until use in the bioassay.

The PROTEINSIMPLE® Simon™ western blotting bioassay assay employs aquantitative western blotting immunoassay technique using an antibodyspecific for occludin to quantitatively detect occludin in the testsamples. Cell samples are lysed and normalized for proteinconcentration. Normalized samples and molecular weight standards arethen loaded and ran on a denatured protein separation gel usingcapillary electrophoresis. The proteins in the gel are then immobilizedand immunoprobed using a primary antibody specific for occludin. Theimmobilized proteins are immunoprobed with an enzyme-linked detectionantibody that binds the primary antibody. A chemiluminescent substratesolution is then added to the immobilized proteins to allowchemiluminescent development in proportion to the amount of occludinbound in the immobilization. The chemiluminescent development can bestopped at a specific time and the intensity of the chemiluminescentsignal can be measured and compared to positive and negative controls.

Keratinocyte Monolayer Permeability: Changes in the permeability of akeratinocyte monolayer due to each of the active ingredients, any one ofthe combination of ingredients, or compositions having said combinationsdisclosed in the specification can be measured. Keratinocyte monolayerpermeability is a measure of skin barrier integrity. Keratinocytemonolayer permeability in treated and non-treated keratinocytes can bedetermined using, as a non-limiting example, the In Vitro VascularPermeability assay by Millipore (ECM642). This assay analyzesendothelial cell adsorption, transport, and permeability. Briefly, adulthuman epidermal keratinocytes from Life Technologies (C-005-5C) can beseeded onto a porous collagen-coated membrane within a collection well.The keratinocytes are then incubated for 24 hours at 37° C. and 5% CO₂in Epilife growth media with calcium from Life Technologies(M-EP-500-CA) supplemented with Keratinocyte Growth Supplement (HKGS)from Life Technologies (S-101-5). This incubation time allows the cellsto form a monolayer and occlude the membrane pores. The media is thenreplaced with fresh media with (test sample) or without (non-treatedcontrol) test compounds/extracts and the keratinocytes are incubated foran additional 48 hours at 37° C. and 5% CO₂. To determine permeabilityof the keratinocyte monolayer after incubation with/without the testcompound/extract, the media is replaced with fresh media containing ahigh molecular weight Fluorescein isothiocyanate (FITC)-Dextran and thekeratinocytes are incubated for 4 hours at 37° C. and 5% CO₂. During the4 hours incubation, FITC can pass through the keratinocytes monolayerand porous membrane into the collection well at a rate proportional tothe monolayer's permeability. After the 4 hour incubation, cellviability and the content of FITC in the collection wells can bedetermined. For the FITC content, the media in the collection well iscollected and fluorescence of the media determined at 480 nm (Em) whenexcited at 520 nm. Percent permeability and percent change in comparisonto the non-treated controls can be determined by the followingequations: Percent Permeability=((Mean Ex/Em of test sample)/Mean Ex/Emuntreated control)*100; Percent Change=Percent Permeability of testsample−Percent Permeability of untreated control.

Production of Hyaluronic Acid: Changes in the production of hyaluronicacid in human dermal fibroblasts due to each of the active ingredients,any one of the combination of ingredients, or compositions having saidcombinations disclosed in the specification can be measured. HA is apolysaccharide involved in stabilization of the structure of the matrixand is involved in providing turgor pressure to tissue and cells. As onenon-limiting example, HA production in treated and non-treated adulthuman dermal fibroblasts (HDFa) cells can be determined using theHyaluronan DuoSet ELISA kit from R&D Systems (DY3614). In this assay,for production of samples, subconfluent HDFa cells from CascadeBiologics (C-13-5C) are incubated at 37° C. and 10% CO₂ in starvationmedium (0.15% fetal bovine serum and 1% Penicillin Streptomycin solutionin Dulbecco's Modified Eagle Medium) for 72 hours prior to treatment.The cells are then incubated with fresh starvation medium with eithertest compound, positive control (phorbol 12-myristate 13-acetate fromSigma-Aldrich (P1585) and platelet derived growth factor fromSigma-Aldrich (P3201)), or no additive for 24 hours. Media is thencollected and frozen at −80° C. until use in the ELISA assay.

Briefly, the ELISA assay employs a quantitative sandwich enzymeimmunoassay technique whereby a capture antibody specific for HA can bepre-coated onto a microplate. Standards and media from treated anduntreated cells are pipetted into the microplate wells to enable any HApresent to be bound by the immobilized antibody. After washing away anyunbound substances, an enzyme-linked detection antibody specific for HAis added to the wells. Following a wash to remove any unboundantibody-enzyme reagent, a substrate solution is added to the wells toallow color development in proportion to the amount of HA bound in theinitial step. The color development is stopped at a specific time andthe intensity of the color at 450 nm can be measured using a microplatereader.

Inhibition of Hyaluronidase Activity: Changes in the activity ofhyaluronidase due to each of the active ingredients, any one of thecombination of ingredients, or compositions having said combinationsdisclosed in the specification can be measured. Hyaluronidase is anenzyme that degrades HA. HA is a polysaccharide involved instabilization of the structure of the matrix and is involved inproviding turgor pressure to tissue and cells. As one non-limitingexample, hyaluronidase activity can be determined using an in vitroprotocol modified from Sigma-Aldrich protocol # EC 3.2.1.35. Briefly,hyaluronidase type 1-S from Sigma-Aldrich (H3506) is added to microplatereaction wells containing test compound or controls. Tannic acid can beused as a positive control inhibitor, no test compound can be added forthe control enzyme, and wells with test compound or positive control butwithout hyaluronidase can be used as a background negative control. Thewells are incubated at 37° C. for 10 minutes before addition ofsubstrate (HA). Substrate is added and the reactions incubated at 37° C.for 45 minutes. A portion of each reaction solution is then transferredto and gently mixed in a solution of sodium acetate and acetic acid pH3.75 to stop that portion of the reaction (stopped wells). The stoppedwells and the reaction wells should both contain the same volume ofsolution after addition of the portion of the reaction solution to thestopped wells. Both the reaction wells and the stopped wells areincubated for 10 minutes at room temperature. Absorbance at 600 nm isthen measured for both the reaction wells and the stopped wells.Inhibition can be calculated using the following formulas: Inhibitor (orcontrol) activity=(Inhibitor stopped wells absorbance at 600nm−inhibitor reaction wells absorbance at 600 nm); Initialactivity=control enzyme absorbance at 600 nm; PercentInhibition=[(Initial activity/Inhibitor Activity)*100]-100.

Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ) Activity:Changes in the activity of PPAR-γ due to each of the active ingredients,any one of the combination of ingredients, or compositions having saidcombinations disclosed in the specification can be measured. PPAR-γ is areceptor critical for the production of sebum. As one non-limitingexample, the activity of PPAR-γ can be determined using a bioassay thatanalyzes the ability of a test compound or composition to inhibitbinding of a ligand. Briefly, fluorescent small-molecule pan-PPARligand, FLUORMONE™ Pan-PPAR Green, available from Life Technologies(PV4894), can be used to determine if test compounds or compositions areable to inhibit binding of the ligand to PPAR-γ. The samples wellsinclude PPAR-γ and fluorescent ligand and either: test compound orcomposition (test); a reference inhibitor, rosiglitazone (positivecontrol); or no test compound (negative control). The wells areincubated for a set period of time to allow the ligand opportunity tobind the PPAR-γ. The fluorescence polarization of each sample well canthen be measured and compared to the negative control well to determinethe percentage of inhibition by the test compound or composition.

Cytokine array: Human epidermal keratinocytes are cultured to 70-80%confluency. The media in the plate is aspirated and 0.025% trypsin/EDTAis added. When the cells became rounded, the culture dish is gentlytapped to release the cells. The trypsin/EDTA containing cells areremoved from the culture dish and neutralized. Cells are centrifuged for5 min. at 180×g to form a pellet of cells. The supernatant is aspirated.The resulting pellet is resuspended in EpiLife™ media (CascadeBiologics). The cells are seeded in 6-well plates at approximately10-20% confluency. After the cells became approximately 80% confluent,the media is aspirated and 1.0 ml of EpiLife™, along with phorbol13-Myristate 12-acetate (“PMA”) (a known inducer of inflammation) andthe test composition dilutions are added to two replicate wells (i.e.,1.0% (100 μl of 100× stock) and 0.1% (10 μl of 100× stock) testcompositions are diluted into a final volume of 1 ml EpiLife GrowthMedium). The media is gently swirled to ensure adequate mixing. Inaddition, 1.0 ml of EpiLife™ is added to the control wells, with andwithout additional PMA. The plates are then incubated at 37±1° C. and5.0±1% CO₂ for approximately 5 hours after dosing. Following this 5-hourincubation, all media is collected in conical tubes and frozen at −70°C.

For analysis, a 16-pad hybridization chamber is attached to 16-pad FASTslides arrayed in triplicate with 16 anti-cytokine antibodies plusexperimental controls (Whatman BioSciences), and the slides are placedinto a FASTFrame (4 slides per frame) for processing. Arrays are blockedfor 15 min. at room temp. using 70 ml S&S Protein Array Blocking buffer(Whatman Schleicher and Scheull). Blocking buffer is removed and 70 mlof each supernatant sample is added to each array. Arrays are incubatedfor 3 hours at room temp. with gentle agitation. Arrays are washed 3times with TBS-T. Arrays are treated with 70 ml of an antibody cocktail,containing one biotinylated antibody corresponding to each of thearrayed capture antibodies. Arrays are incubated for 1 hour at roomtemp. with gentle agitation. Arrays are washed 3 times with TBS-T.Arrays are incubated with 70 ml of a solution containingstreptavidin-Cy5 conjugate for 1 hour at room temp. with gentleagitation. Arrays are washed 3 times with TBS-T, quickly rinsed inde-ionized water, and dried.

Slides can be imaged in a Perkin-Elmer ScanArray 4000 confocalfluorescent imaging system. Array images can be saved and analyzed usingImaging Research ArrayVision software. Briefly, spot intensities aredetermined by subtracting background signal. Spot replicates from eachsample condition can be averaged and then compared to the appropriatecontrols.

Endothelial Tube Formation: Endothelial tube formation is involved inangiogenesis and micro-vessel capillary formation. Capillary formationand angiogenesis may contribute to redness and rosacea of the skin. Theability for endothelial cells to form tubes in the presence or absenceof test extracts and compounds may be determined using a capillarytubule disruption assay with pre-formed primary human umbilical veinendothelial cells (HUVEC) in a cell culture system.

Briefly, HUVECs are cultured in vitro on Extracellular Matrix, whichstimulates the attachment and tubular morphogenesis of endothelial cellsto form capillary-like lumen structures. These in vitro formed capillarytubules are similar to human blood vessel capillaries in many aspects.The capillary tube assay is based on this phenomenon and is used forevaluation of potential vasculature targeting agents.

HUVEC cultures are grown in a 5% CO₂ 37° C. cell incubator. The fullgrowth medium for HUVECs is Endothelial Cell Basal Medium (EBM)supplemented with 2% fetal bovine serum (FBS), 12 μg/ml bovine brainextract, 1 μg/ml hydrocortisone, and 1 μg/ml GA-1000(gentamicin-amphothericin). HUVEC cultures between passage 3 and 8 maybe used for all assay experiments.

HUVECs are pre-labeled with fluorescent agent Calcein AM and seeded inExtracellular Matrix coated 96-well culture plate with their full growthmedium. After about four hours of the morphogenesis process, theendothelial capillary tubes should be formed. Then, test agent indesigned doses in 50 μl volume is applied into the formed capillarytubule cultures as treatment conditions. The no-treatment controls canbe added with vehicle of test agents. Sutent, a FDA approvedanti-angiogenic drug one concentration can be included as assayperformance control. After about six hours of treatment, the endothelialtubule morphology in each well is examined by microscopy, imaged, andthe capillary disrupting activities under treatment conditions can bequantitatively analyzed. Each test conditions can be conducted induplicate wells, including controls.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   Cosmetic Ingredient Dictionary, Third Edition, CTFA, 1982-   International Cosmetic Ingredient Dictionary, Fourth edition, CTFA,    1991-   International Cosmetic Ingredient Dictionary and Handbook, Tenth    Edition, CTFA, 2004-   International Cosmetic Ingredient Dictionary and Handbook, Twelfth    Edition, CTFA, 2008

1. A method for reducing a presence of sebum on skin, reducing anappearance of shiny skin, or reducing an appearance of the size of skinpores, the method comprising topically applying to skin a compositioncomprising: a navy bean extract comprising amino acids; an extract ofBambusa vulgaris wendl shoots; and charcoal; and wherein topicalapplication of the composition to the skin reduces the presence of sebumon the skin, reduces the appearance of shiny skin, or reduces theappearance of the size of the skin pores.
 2. The method of claim 1,wherein the composition comprises: 0.01% to 5% by weight of navy beanextract comprising amino acids; 0.01% to 5% by weight of the extract ofBambusa vulgaris wendl shoots; and 0.01% to 5% charcoal.
 3. The methodof claim 1, wherein the composition further comprises: kaolin; andbentonite.
 4. The method of claim 3, wherein the composition comprises:20% to 35% by weight kaolin; and 1% to 8% by weight bentonite.
 5. Themethod of claim 1, wherein the composition further comprises: water;glycerin; propylene glycol; iron oxide; butylene glycol; andphenoxyethanol.
 6. The method of claim 5, wherein the compositioncomprises about: 47% by weight of water; 7% by weight of glycerin; 5% byweight of propylene glycol; 1.2% by weight of iron oxide; 0.7% by weightof butylene glycol; and 0.6% by weight of phenoxyethanol.
 7. The methodof claim 1, wherein the composition further comprises water.
 8. Themethod of claim 1, wherein the composition is a gel.
 9. The method ofclaim 1, wherein the composition is an oil-in-water emulsion.
 10. Themethod of claim 1, wherein the composition remains on the skin for atleast 10 minutes and is then removed from the skin.
 11. The method ofclaim 1, wherein the skin is oily skin.
 12. The method of claim 1,wherein the composition is a facial mask that is applied to facial skin.